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Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T 50 , a 725% increase in half-life, a 5.5°C increase in T m , and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase., (© 2024 National Research Council Canada and The Authors. Biotechnology and Bioengineering by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)

Curcumin is a plant-derived secondary metabolite exhibiting antitumor, neuroprotective, antidiabetic activities, and so on. We previously isolated Escherichia coli as an enterobacterium exhibiting curcumin-converting activity from human feces, and discovered an enzyme showing this activity (CurA) and named it NADPH-dependent curcumin/dihydrocurcumin reductase. From soil, here, we isolated a curcumin-degrading microorganism (No. 34) using the screening medium containing curcumin as the sole carbon source and identified as Rhodococcus sp. A curcumin-degrading enzyme designated as CurH was purified from this strain and characterized, and compared with CurA. CurH catalyzed hydrolytic cleavage of a carbon-carbon bond in the β-diketone moiety of curcumin and its analogs, yielding two products bearing a methyl ketone terminus and a carboxylic acid terminus, respectively. These findings demonstrated that a curcumin degradation reaction catalyzed by CurH in the soil environment was completely different from the one catalyzed by CurA in the human microbiome. Of all the curcumin analogs tested, suitable substrates for the enzyme were curcuminoids (i.e., curcumin and bisdemethoxycurcumin) and tetrahydrocurcuminoids. Thus, we named this enzyme curcuminoid hydrolase. The deduced amino acid sequence of curH exhibited similarity to those of members of acetyl-CoA C-acetyltransferase family. Considering results of oxygen isotope analyses and a series of site-directed mutagenesis experiments on our enzyme, we propose a possible catalytic mechanism of CurH, which is unique and distinct from those of enzymes degrading β-diketone moieties such as β-diketone hydrolases known so far., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60-99%) and high selectivities (ee > 95%). The best bioreductions-in terms of conversion (>90%) and enantiomeric excess (>99% ee)-at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions.

Propanil residues can contaminate habitats where microbial degradation is predominant. In this study, an efficient propanil-degrading strain C-1 was isolated from paddy and identified as Rhodococcus sp. It can completely degrade 10 μg/L-150 mg/L propanil within 0.33-10 h via the hydrolysis of the amide bond, forming 3,4-dichloroaniline. A novel bifunctional amidase, PamC, was identified in strain C-1. PamC can catalyze the hydrolysis of the amide bond of propanil to produce 3,4-dichloroaniline as well as the hydrolysis of the ester bonds of aryloxyphenoxypropionate herbicides (APPHs, clodinafop-propargyl, cyhalofop-butyl, fenoxaprop- p -ethyl, fluazifop- p -butyl, haloxyfop- p -methyl, and quizalofop -p -ethyl) to form aryloxyphenoxypropionic acids. Molecular docking and site-directed mutagenesis confirmed that the catalytic triad Lys82-Ser157-Ser181 was the active center for PamC to hydrolyze propanil and cyhalofop-butyl. This study presents a novel bifunctional amidase with capabilities for both amide and ester bond hydrolysis and enhances our understanding of the molecular mechanisms underlying the degradation of propanil and APPHs.

Phthalate esters (PAEs) are widely used as plasticizers and cause serious complex pollution problem in environment. Thus, strains with efficient ability to simultaneously degrade various PAEs are required. In this study, a newly isolated strain Rhodococcus sp. AH-ZY2 can degrade 500 mg/L Di-n-octyl phthalate completely within 16 h and other 500 mg/L PAEs almost completely within 48 h at 37 °C, 180 rpm, and 2 % (v/v) inoculum size of cultures with a OD 600 of 0.8. OD 600 = 0.8, 2 % (v/v). Twenty genes in its genome were annotated as potential esterase and four of them (3963, 4547, 5294 and 5359) were heterogeneously expressed and characterized. Esterase 3963 and 4547 is a type I PAEs esterase that hydrolyzes PAEs to phthalate monoesters. Esterase 5294 is a type II PAEs esterase that hydrolyzes phthalate monoesters to phthalate acid (PA). Esterase 5359 is a type III PAEs esterase that simultaneously degrades various PAEs to PA. Molecular docking results of 5359 suggested that the size and indiscriminate binding feature of spacious substrate binding pocket may contribute to its substrate versatility. AH-ZY2 is a potential strain for efficient remediation of PAEs complex pollution in environment. It is first to report an esterase that can efficiently degrade mixed various PAEs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry., Methods and Results: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt., Conclusion: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Isoamyl fatty acid esters (IAFEs) are widely used as fruity flavor compounds in the food industry. In this study, various IAFEs were synthesized from isoamyl alcohol and various fatty acids using a cutinase enzyme (Rcut) derived from Rhodococcus bacteria. Rcut was immobilized on methacrylate divinylbenzene beads and used to synthesize isoamyl acetate, butyrate, hexanoate, octanoate, and decanoate. Among them, Rcut synthesized isoamyl butyrate (IAB) most efficiently. Docking model studies showed that butyric acid was the most suitable substrate in terms of binding energy and distance from the active site serine (Ser114) γ-oxygen. Up to 250 mM of IAB was synthesized by adjusting reaction conditions such as substrate concentration, reaction temperature, and reaction time. When the enzyme reaction was performed by reusing the immobilized enzyme, the enzyme activity was maintained at least six times. These results demonstrate that the immobilized Rcut enzyme can be used in the food industry to synthesize a variety of fruity flavor compounds, including IAB.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP + or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD + , NADP + , flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N 5 OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O 2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

Cholesterol oxidases (ChOxes) are enzymes that catalyze the oxidation of cholesterol to cholest-4-en-3-one. These enzymes find wide applications across various diagnostic and industrial settings. In addition, as a pathogenic factor of several bacteria, they have significant clinical implications. The current classification system for ChOxes is based on the type of bond connecting FAD to the apoenzyme, which does not adequately illustrate the enzymatic and structural characteristics of these proteins. In this study, we have adopted an integrative approach, combining evolutionary analysis, classic enzymatic techniques and computational approaches, to elucidate the distinct features of four various ChOxes from Rhodococcus sp. (RCO), Cromobacterium sp. (CCO), Pseudomonas aeruginosa (PCO) and Burkhoderia cepacia (BCO). Comparative and evolutionary analysis of substrate-binding domain (SBD) and FAD-binding domain (FBD) helped to reveal the origin of ChOxes. We discovered that all forms of ChOxes had a common ancestor and that the structural differences evolved later during divergence. Further examination of amino acid variations revealed SBD as a more variable compared to FBD independently of FAD coupling mechanism. Revealed differences in amino acid positions turned out to be critical in determining common for ChOxes properties and those that account for the individual differences in substrate specificity. A novel look with the help of chemical descriptors on found distinct features were sufficient to attempt an alternative classification system aimed at application approach. While univocal characteristics necessary to establish such a system remain elusive, we were able to demonstrate the substrate and protein features that explain the differences in substrate profile., Competing Interests: Declaration of competing interest The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. The authors whose names are listed immediately below report the following details of affiliation or involvement in an organization or entity with a financial or non-financial interest in the subject matter or materials discussed in this manuscript. Please specify the nature of the conflict on a separate sheet of paper if the space below is inadequate. This statement is signed by all the authors to indicate agreement that the above information is true and correct (a photocopy of this form may be used if there are more than 10 authors)., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii ( Rjo GlgAb and Rjo GlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. Rjo GlgAb and Rjo GlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, Rjo GlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. Rjo GlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, Rjo GlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, Rjo GlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O 2 ) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components., (Copyright © 2024. Published by Elsevier Inc.)

Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Plastic waste management has become a global issue. Polyethylene (PE) is the most abundant synthetic plastic worldwide, and one of the most resistant to biodegradation. Indeed, few bacteria can degrade polyethylene. In this paper, the transcriptomic analysis unveiled for the first time Rhodococcus opacus R7 complex genetic system based on diverse oxidoreductases for polyethylene biodegradation. The RNA-seq allowed uncovering genes putatively involved in the first step of oxidation. In-depth investigations through preliminary bioinformatic analyses and enzymatic assays on the supernatant of R7 grown in the presence of PE confirmed the activation of genes encoding laccase-like enzymes. Moreover, the transcriptomic data allowed identifying candidate genes for the further steps of short aliphatic chain oxidation including alkB gene encoding an alkane monooxygenase, cyp450 gene encoding cytochrome P450 hydroxylase, and genes encoding membrane transporters. The PE biodegradative system was also validated by FTIR analysis on R7 cells grown on polyethylene., (© 2021. The Author(s).)

para -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via the "hydroquinone (HQ) pathway" in Gram-negative degraders, whereas it proceeds via the "benzenetriol (BT) pathway" in Gram-positive ones. The "HQ pathway" is initiated by a single-component PNP 4-monooxygenase and the "BT pathway" by a two-component PNP 2-monooxygenase. Their regioselectivity intrigued us enough to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis strain RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of the group D flavin-dependent monooxygenases with an acyl coenzyme A (acyl-CoA) dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate toward flavin adenine dinucleotide (FAD), hence positioning the substrate properly. An N450A variant was found with improved activity for 4NC and 2C4NP-probably because of the reduced steric hindrance. PnpA1 shows an obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be caused by the unique Thr296 and a different conformation in the loop from positions 449 to 454 (loop 449-454). Above all, our study allows structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regioselectivity was proposed: the different PNP binding manners determine their choice of ortho - or para -hydroxylation on PNP. IMPORTANCE Single-component PNP monoxygenases hydroxylate PNP at the 4 position, while two-component ones do so at the 2 position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed, and a variant with improved activities toward 4NC and 2C4NP was obtained. Moreover, through comparison of the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at the molecular level. In addition, these results will also be of further aid in rational design of enzymes in bioremediation and biosynthesis.

Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)-camphor is available from natural sources. Optically pure (1S)-camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from α-pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolyzed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol, borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from α-pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side-product., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2'-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO 2 ) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants having health hazards. PAH-utilizing bacterial strains were isolated from petroleum-contaminated soil from siding area, Bijwasan supply location of BPCL, Delhi, India. Bacterial strains with different morphology were isolated and acclimatized to a mixture of low molecular weight PAH compounds in the concentration range of 50-10,000 mg/L. Two bacterial strains surviving at 10,000 mg/L PAH concentration were identified as Kocuria flava and Rhodococcus pyridinivorans, based on 16S rRNA gene sequencing and phylogenetic analysis over MEGA X, are reported for the first time for PAH degradation. The strain K. flava could degrade phenanthrene, anthracene, and fluorene with efficiency of 55.13%, 59.01%, and 63.46%, whereas R. pyridinivorans exhibited 62.03%, 64.99%, and 66.79% degradation for respective PAHs at initial PAH concentration of 10 mg/L. Slightly lower degradation of phenanthrene could be attributed to its more stable chemical structure. The consortium of both the strains degraded 61.32%, 64.72%, and 66.64%, of 10 mg/L of phenanthrene, anthracene, and fluorene, respectively, in 15 days of incubation period indicating no synergistic or antagonistic effect towards degradation. Catechol 2,3-dioxygenase (C23O), dehydrogenase and peroxidase enzyme activities during PAH degradation coincided with degradation of PAHs, thus highlighting the role of these enzymes in catabolising three-ring PAHs. This is the first investigation confirming the participation of C23O, dehydrogenase and peroxidases enzyme profiles throughout the period of degradation. The study concludes that these strains can play significant role in microbial remediation of PAH-contaminated environment., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: 3-Ketosteroid Δ 1 -dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity., Results: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔG bind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi K mA  = 23.7 μM) than to AD (K mA  = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (k cat /K mA  = 9.25∙10 6  M -1  s -1 ) is almost 20 times higher than measured for KSTD1 (k cat /K mA  = 4.71∙10 5  M -1  s -1 )., Conclusions: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99 % ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93 % ee)., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown., Results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCD B2 ), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthR B2 ), a ferredoxin reductase (EthA B2 ), a cytochrome P450 monooxygenase (EthB B2 ), a ferredoxin (EthC B2 ) and a 10-kDa protein of unknown function (EthD B2 ). Complementation with EthABCD B2 and EthABD B2 , but not EthABC B2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCD B2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCD B2 or EthABD B2 but not EthABC B2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD B2 acted as a ferredoxin in strain B2. EthABD B2 displayed maximal activity at 30 °C and pH 7.5., Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

Machine learning (ML) has pervaded most areas of protein engineering, including stability and stereoselectivity. Using limonene epoxide hydrolase as the model enzyme and innov'SAR as the ML platform, comprising a digital signal process, we achieved high protein robustness that can resist unfolding with concomitant detrimental aggregation. Fourier transform (FT) allows us to take into account the order of the protein sequence and the nonlinear interactions between positions, and thus to grasp epistatic phenomena. The innov'SAR approach is interpolative, extrapolative and makes outside-the-box, predictions not found in other state-of-the-art ML or deep learning approaches. Equally significant is the finding that our approach to ML in the present context, flanked by advanced molecular dynamics simulations, uncovers the connection between epistatic mutational interactions and protein robustness., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The success of members of the genus Rhodococcus in colonizing arid rocky environments is owed in part to desiccation tolerance and an ability to extract iron through the secretion and uptake of siderophores. Here, we report a comprehensive genomic and taxonomic analysis of Rhodococcus qingshengii strain S10 isolated from eathered serpentine rock at the arid Khalilovsky massif, Russia. Sequence comparisons of whole genomes and of selected marker genes clearly showed strain S10 to belong to the R. qingshengii species. Four prophage sequences within the R. qingshengii S10 genome were identified, one of which encodes for a putative siderophore-interacting protein. Among the ten non-ribosomal peptides synthase (NRPS) clusters identified in the strain S10 genome, two show high homology to those responsible for siderophore synthesis. Phenotypic analyses demonstrated that R. qingshengii S10 secretes siderophores and possesses adaptive features (tolerance of up to 8% NaCl and pH 9) that should enable survival in its native habitat within dry serpentine rock.

The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C-H bond in methyl groups need to be activated.

Backgound: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy., Methods: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (k cat /K m ) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2., Results: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules., Conclusions: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications., General Significance: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gentisate 1,2-dioxygenases belong to the class III ring-cleaving dioxygenases catalyzing key reactions of aromatic compounds degradation by aerobic microorganisms. In the present work, the results of complete molecular, structural, and functional investigations of the gentisate 1,2-dioxygenase (rho-GDO) from a gram-positive bacterium Rhodococcus opacus 1CP growing on 3-hydroxybenzoate as a sole source of carbon and energy are presented. The purified enzyme showed a narrow substrate specificity. Among fourteen investigated substrate analogues only gentisate was oxidized by the enzyme, what can be potentially applied in biosensor technologies. The rho-GDO encoding gene was identified in the genomic DNA of the R. opacus 1CP. According to phylogenetic analysis, the rho-GDO belongs to the group of apparently most recently acquired activities in bacterial genera Rhodococcus, Arthrobacter, Corynebacterium, Nocardia, Amycolatopsis, Comamonas, and Streptomyces. Homology modeling the rho-GDO 3D-structure demonstrates the composition identity of the first-sphere residues of the active site of rho-GDO and salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans (RCSB PDB: 2PHD), despite of their different substrate specificities. The phenomenon described for the first time for this family of enzymes supposes a more complicated mechanism of substrate specificity than previously imagined, and makes the rho-GDO a convenient model for a novel direction of structure-function relationship studies., Competing Interests: Declaration of competing interest The authors declare that they have not known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All authors have not and will have not any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Two genes (choRI and choRII) encoding cholesterol oxidases belonging to the vanillyl-alcohol oxidase (VAO) family were cloned on the basis of putative cholesterol oxidase gene sequences in the genome sequence data of Rhodococcus erythropolis PR4. The genes corresponding to the mature enzymes were cloned in a pET vector and expressed in Escherichia coli. The two cholesterol oxidases produced from the recombinant E. coli were purified to examine their properties. The amino acid sequence of ChoRI showed significant similarity (57%) to that of ChoRII. ChoRII was more stable than ChoRI in terms of pH and thermal stability. The substrate specificities of these enzymes differed distinctively from one another. Interestingly, the activities of ChoRII toward β-cholestanol, β-sitosterol, and stigmasterol were 2.4-, 2.1-, and 1.7-fold higher, respectively, than those of cholesterol. No cholesterol oxidases with high activity toward these sterols have been reported so far. The cholesterol oxidation products from these two enzymes also differed. ChoRI and ChoRII oxidized cholesterol to form cholest-4-en-3-one and 6β-hydroperoxycholest-4-en-3-one, respectively., Competing Interests: The authors have declared that no competing interests exist.

The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4-phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4-phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O -demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA , encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta -cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC , encoding the meta -cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL , encoding a CYP255A2 family P450 and an ortho -cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O -demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities ( k cat / K M ): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoA EP4 's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin., Competing Interests: The authors declare no competing interest.

As self-subunit swapping chaperones or metallochaperones, the activators assist nitrile hydratases to take up metal ions and they are essential for active expression of nitrile hydratases. Compared with nitrile hydratases, the activators have a low sequence identity. Study of the activation characteristics and the relationships between structures and functions of the activators is of great significance for understanding the maturation mechanism of nitrile hydratase. We co-expressed low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 with four heterologous activators respectively and determined their activation abilities. Then we made sequence analysis and structure modelling, and studied the functions of the important domains of the activators. Results showed that all four heterologous activators could activate L-NHase, however, the specific activities of L-NHases were different after activation. L-NHase showed the highest specific activity after being activated by activator A, which was 97.79% of that of the original enzyme, but the specific activity of L-NHase after being activated by activator G was only 23.94% of that of the original enzyme. Activator E and activator G had conserved domains (TIGR03889), and deletion of their partial sequences resulted in a substantial loss of activation abilities for both activators. Replacing the N-terminal sequence of activator G with the N-terminal sequence of activator E, and adding the C-terminal sequence of activator E to the C-terminus of activator G could increase the specific activity of L-NHase by 178.40%. The activation by nitrile hydratase activators was universal and specific, and the conserved domains of activators were critical for activation, while the N-terminal domain and C-terminal domain also had important effects on activation.

The efficient immobilization of haloalkane dehalogenase (DhaA) on carriers with retaining of its catalytic activity is essential for its application in environmental remediation. In this work, adsorption orientation and conformation of DhaA on different functional surfaces were investigated by computer simulations; meanwhile, the mechanism of varying the catalytic activity was also probed. The corresponding experiments were then carried out to verify the simulation results. (The simulations of DhaA on SAMs provided parallel insights into DhaA adsorption in carriers. Then, the theory-guided experiments were carried out to screen the best surface functional groups for DhaA immobilization.) The electrostatic interaction was considered as the main impact factor for the regulation of enzyme orientation, conformation, and enzyme bioactivity during DhaA adsorption. The synergy of overall conformation, enzyme substrate tunnel structural parameters, and distance between catalytic active sites and surfaces codetermined the catalytic activity of DhaA. Specifically, it was found that the positively charged surface with suitable surface charge density was helpful for the adsorption of DhaA and retaining its conformation and catalytic activity and was favorable for higher enzymatic catalysis efficiency in haloalkane decomposition and environmental remediation. The neutral, negatively charged surfaces and positively charged surfaces with high surface charge density always caused relatively larger DhaA conformation change and decreased catalytic activity. This study develops a strategy using a combination of simulation and experiment, which can be essential for guiding the rational design of the functionalization of carriers for enzyme adsorption, and provides a practical tool to rationally screen functional groups for the optimization of adsorbed enzyme functions on carriers. More importantly, the strategy is general and can be applied to control behaviors of different enzymes on functional carrier materials.

The non-hydrolytic ring opening of 1,2-epoxides in the presence of limonene epoxide hydrolases (LEHs) and different nucleophiles has been investigated. Lyophilized, wild-type LEHs were tested in selected water-saturated organic solvents in the presence of cyclohexene oxide as substrate and different alcohols, thiols and primary amines as nucleophiles. Although the LEHs retained an appreciable catalytic activity under different reaction conditions, formation of the desired 1,2-substituted cyclohexanols was not observed. Alternatively, LEH variants incapable of performing the hydrolytic reaction were generated by site-directed mutagenesis and tested in aqueous media in the presence of different water-soluble nucleophiles and cyclohexene oxide. Under defined reaction conditions, an acceleration of up to about threefold of the spontaneous reaction rate was observed in the presence of sodium azide and potassium thiocyanate as nucleophiles., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD + -dependent oxidation of 10-hydroxystearic acid.

N -acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA , which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB ) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency ( k cat /K M ) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 × 10 6 to 1.45 × 10 6 M -1 s -1 , with distinctly low K M values (0.16 - 0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.

Microbial degradation is considered to be the most acceptable method for degradation of chlorimuron-ethyl, a typical long-term residual sulfonylurea herbicide, but the underlying mechanism at the genetic and biochemical levels is unclear. In this work, the genome sequence of the chlorimuron-ethyl-degrading bacterium Rhodococcus erythropolis D310-1 was completed, and the gene clusters responsible for the degradation of chlorimuron-ethyl in D310-1 were predicted. A carboxylesterase gene, carE, suggested to be responsible for carboxylesterase de-esterification, was cloned from D310-1. CarE was expressed in Escherichia coli BL21 and purified to homogeneity. The active site of the chlorimuron-ethyl-degrading enzyme CarE and the biochemical activities of CarE were elucidated. The results demonstrated that CarE is involved in catalyzing the de-esterification of chlorimuron-ethyl. A carE deletion mutant strain, D310-1ΔcarE, was constructed, and the chlorimuron-ethyl degradation rate in the presence of 100 mg L -1 chlorimuron-ethyl within 120 h decreased from 86.5 % (wild-type strain D310-1) to 58.2 % (mutant strain D310-1ΔcarE). Introduction of the plasmid pNit-carE restored the ability of the mutant strain to utilize chlorimuron-ethyl. This study is the first to demonstrate that carboxylesterase can catalyze the de-esterification reaction of chlorimuron-ethyl and provides new insights into the mechanism underlying the degradation of sulfonylurea herbicides and a theoretical basis for the utilization of enzyme resources., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19 F NMR in conjunction with fluorinated substrate analogs to directly measure p K a values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the p K a of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19 F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the p K a of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C 2 component of HPAH. 19 F NMR also revealed a p K a of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C 2 , respectively, are consistent with p K a tuning by one or more H-bonding interactions. Our implementation of 19 F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms., (© 2020 Pitsawong et al.)

Quantification of polyamines, including putrescine, is generally performed using high-performance liquid chromatography (HPLC) or gas chromatography. However, these methods are time-consuming because of sample derivatization and analytical reagent preparation. In this study, we developed a simple and high-throughput putrescine quantification method on a 96-well microtiter plate using putrescine oxidase from Rhodococcus erythropolis NCIMB 11540, peroxidase, 4-aminoantipyrine, and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt. The developed method (named as PuO-POD-4AA-TOPS method) was applicable to bacterial culture supernatants. Furthermore, putrescine concentrations determined by the developed method roughly corresponded to the concentrations determined by HPLC., (Copyright © 2020 Elsevier Inc. All rights reserved.)

For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones Rr GroEL and Rr GroES from Rhodococcus ruber , a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber . In the E. coli host, fusion expression of nitrilase with either Rr GroES or Rr GroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32-47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of Rr GroES or Rr GroEL with a plasmid, the engineered strains R. ruber TH3 dNHase:: Rr Nit (pNV18.1-P ami - Rr Nit-P ami - Rr GroES) and TH3 dNHase:: Rr Nit (pNV18.1-P ami - Rr Nit-P ami - Rr GroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the Rr GroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-P ami - Rr Nit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts.

Diazo compounds are versatile reagents in chemical synthesis and biology due to the tunable reactivity of the diazo functionality and its compatibility with living systems. Much effort has been made in recent years to explore their accessibility and synthetic potential; however, their preparation through stereoselective enzymatic asymmetric synthesis has been scarcely reported in the literature. Alcohol dehydrogenases (ADHs, also called ketoreductases, KREDs) are powerful redox enzymes able to reduce carbonyl compounds in a highly stereoselective manner. Herein, we have developed the synthesis and subsequent bioreduction of nine α-diazo-β-keto esters to give optically active α-diazo-β-hydroxy esters with potential applications as chiral building blocks in chemical synthesis. Therefore, the syntheses of prochiral α-diazo-β-keto esters bearing different substitution patterns at the adjacent position of the ketone group (N 3 CH 2 , ClCH 2 , BrCH 2 , CH 3 OCH 2 , NCSCH 2 , CH 3 , and Ph) and in the alkoxy portion of the ester functionality (Me, Et, and Bn), were carried out through the diazo transfer reaction to the corresponding β-keto esters in good to excellent yields (81-96%). After performing the chemical reduction of α-diazo-β-keto esters with sodium borohydride and developing robust analytical conditions to monitor the biotransformations, their bioreductions were exhaustively studied using in-house made Escherichia coli overexpressed and commercially available KREDs. Remarkably, the corresponding α-diazo-β-hydroxy esters were obtained in moderate to excellent conversions (60 to >99%) and high selectivities (85 to >99% ee ) after 24 h at 30 °C. The best biotransformations in terms of conversion and enantiomeric excess were successfully scaled up to give the expected chiral alcohols with almost the same activity and selectivity values observed in the enzyme screening experiments.

All nitroarene dioxygenases reported so far originated from Nag-like naphthalene dioxygenase of Gram-negative strains, belonging to group III of aromatic ring-hydroxylating oxygenases (RHOs). Gram-positive Rhodococcus sp. strain ZWL3NT utilizes 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. It was also reported that 3NT degradation was constitutive and the intermediate was 3-methylcatechol. In this study, a gene cluster ( bndA1A2A3A4 ) encoding a multicomponent dioxygenase, belonging to group IV of RHOs, was identified. Recombinant Rhodococcus imtechensis RKJ300 carrying bndA1A2A3A4 exhibited 3NT dioxygenase activity, converting 3NT into 3-methylcatechol exclusively, with nitrite release. The identity of the product 3-methylcatechol was confirmed using liquid chromatography-mass spectrometry. A time course of biotransformation showed that the 3NT consumption was almost equal to the 3-methylcatechol accumulation, indicating a stoichiometry conversion of 3NT to 3-methylcatechol. Unlike reported Nag-like dioxygenases transforming 3NT into 4-methylcatechol or both 4-methylcatechol and 3-methylcatechol, this Bph-like dioxygenase (dioxygenases homologous to the biphenyl dioxygenase from Rhodococcus sp. strain RHA1) converts 3NT to 3-methylcatechol without forming 4-methylcatechol. Furthermore, whole-cell biotransformation of strain RKJ300 with bndA1A2A3A4 and strain ZWL3NT exhibited the extended and same substrate specificity against a number of nitrobenzene or substituted nitrobenzenes, suggesting that BndA1A2A3A4 is likely the native form of 3NT dioxygenase in strain ZWL3NT. IMPORTANCE Nitroarenes are synthetic molecules widely used in the chemical industry. Microbial degradation of nitroarenes has attracted extensive attention, not only because this class of xenobiotic compounds is recalcitrant in the environment but also because the microbiologists working in this field are curious about the evolutionary origin and process of the nitroarene dioxygenases catalyzing the initial reaction in the catabolism. In contrast to previously reported nitroarene dioxygenases from Gram-negative strains, which originated from a Nag-like naphthalene dioxygenase, the 3-nitrotoluene (3NT) dioxygenase in this study is from a Gram-positive strain and is an example of a Bph-like nitroarene dioxygenase. The preference of hydroxylation of this enzyme at the 2,3 positions of the benzene ring to produce 3-methylcatechol exclusively from 3NT is also a unique property among the studied nitroarene dioxygenases. These findings will enrich our understanding of the diversity and origin of nitroarene dioxygenase in microorganisms., (Copyright © 2020 American Society for Microbiology.)

Acrylamide is an important bulk chemical used for producing polyacrylamide, which is widely applied in diverse fields, such as enhanced oil recovery and water treatment. Acrylamide production with a superior biocatalyst, free-resting Rhodococcus cells containing nitrile hydratase (NHase), has been proven to be simple but effective, thereby becoming the main method adopted in industry to date. Under the harsh industrial conditions, however, NHase-containing Rhodococcus cells in a natural state are prone to deactivation. Thus, multiple genetic strategies able to evolve recombinant Rhodococcus biocatalysts at either the enzyme or cell level have been reported. While most of the methods on enzyme engineering concentrate on NHase stability enhancement by strengthening the flexible sites, Rhodococcus cell engineering with various methods can enhance both the NHase activity and stability as well. Developing some new types of reactors, especially the microreactor, is also an effective way to improve the hydration process efficiency. Compared with the conventional stirred tank reactor, the membrane dispersion microreactor can enhance the heat and mass transfer in the hydration process with Rhodococcus cells as biocatalysts, thereby significantly improving the productivity of the acrylamide bioproduction process.

The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to large (325 Da) were evaluated. Larger compounds included those with a bi-aryl axis, prepared by the Suzuki coupling reaction, Morita-Baylis-Hillman adducts, heteroatom-linked diarylpyridines prepared by Buchwald-Hartwig cross-coupling reactions and imidazo[1,2- a ]pyridines prepared by the Groebke-Blackburn-Bienaymé multicomponent reaction. The enzyme active site was moderately accommodating, accepting almost all of the small aromatic nitriles, the diarylpyridines and most of the bi-aryl compounds and Morita-Baylis-Hillman products but not the Groebke-Blackburn-Bienaymé products. Nitrile conversion was influenced by steric hindrance around the cyano group, the presence of electron donating groups (e.g., methoxy) on the aromatic ring, and the overall size of the compound.

A thiamine diphosphate-dependent enzyme annotated as a benzoylformate decarboxylase is encoded by gene cluster ro02984-ro02986 in Rhodococcus jostii RHA1 previously shown to generate vanillin and 4-hydroxybenzaldehyde from lignin oxidation, and a closely related gene cluster is also found in the genome of Pseudomonas fluorescens Pf-5. Two hypotheses for possible pathways involving a thiamine diphosphate-dependent cleavage, either C-C cleavage of a ketol or diketone aryl C 3 substrate or decarboxylation of an aryl C 2 substrate, were investigated by expression and purification of the recombinant enzymes and expression of dehydrogenase and oxidase enzymes also found in the gene clusters. The ThDP-dependent enzymes showed no activity for cleavage of aryl C 3 ketol or diketone substrates but showed activity for decarboxylation of benzoylformate and 4-hydroxybenzoylformate. A flavin-dependent oxidase encoded by gene ro02984 was found to oxidize either mandelic acid or phenylglyoxal. The crystal structure of the P. fluorescens decarboxylase enzyme was determined at 1.69 Å resolution, showing similarity to structures of known benzoylformate decarboxylase enzymes. The P. fluorescens decarboxylase enzyme showed enhanced carboligase activity between vanillin and acetaldehyde, rationalized by the presence of alanine versus serine at residue 73 in the enzyme active site, which was investigated further by site-directed mutagenesis of this residue. A hypothesis for a pathway for degradation of aryl C 2 fragments arising from oxidative cleavage of phenylcoumaran and diarylpropane structures in lignin is proposed.

Endoglycoceramidases (EGCs) are family 5 glycoside hydrolases that catalyze hydrolysis of the glycosidic linkages between the oligosaccharide and ceramide moieties of glycosphingolipids. Three orthologs of EGCs, each with distinct substrate specificity, have been identified to date, including EGC-I, EGC-II, and EGALC. Although the structures of EGC-I and EGC-II have been reported, the substrate preference mechanism of EGC enzymes remains unclear. Here, we determined the crystal structure of EGALC from Rhodococcus hoagii 103S at a resolution of 1.20 Å. Distinct from EGC-I and EGC-II, which both have a tunnel-like substrate binding site, the structure of EGALC accommodates substrates in a long groove. Further, the oligosaccharide-binding region of groove could be divided into two small pockets that separately bind to the Gal1 and to the Gal3/Gla3 present in 6-gala series substrates. Structural and sequence comparisons of EGC enzymes revealed that the conformation and length of their Nβ8-Lα1 regions are crucial in determining the architectures of their specific substrate binding sites. Importantly, molecular docking analyses indicate that the substrate specificity of each EGC is mainly derived from the complementarity of its active site groove/tunnel with substrates adopting particular conformations. Our study provide insights for understanding the catalytic mechanism of EGALC, which will help protein engineering for improving the substrate preference and catalytic efficiency of EGC enzymes toward important glycosphingolipid substrates., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Diguanylate cyclases (DGCs) were responsible for the synthesis of second messenger cyclic di-guanosine monophosphate (c-di-GMP), which were involved in various physiological activities of bacterial species. Here, a full-length DGC from Rhodococcus ruber SD3 fused with glutathione-S-transferase (GST) was expressed in E. coli and purified by glutathione agarose resin. The apparent molecular mass of one subunit of the purified diguanylate cyclase with GST tag (GST-DGC) was estimated to be 71.9 kDa by SDS-PAGE, which was approximately in accordance with the theoretical value of 73.0 kDa. The sequence of GST-DGC was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The blue native PAGE indicated that GST-DGC formed octamer. The optimum pH and temperature for GST-DGC activity were 8.0 and 47 °C, respectively. The fusion protein exhibited high thermostability, and 94% of activity was retained when the protein was incubated at 87 °C for 1 h. Moreover, the fusion protein showed pH stability. The Km, Vmax and Kcat values for GST-DGC enzyme were 9.8 μM, 0.7 μM/min and 1.3 S -1 . Some ions such as Zn 2+ , Mn 2+ , Fe 2+ , Ni 2+ and Co 2+ had inhibitory effects on the activity of the protein, while other ions such as Mg 2+ , K + and Na + slightly activated the protein. The fusion protein also showed rather high stability in the presence of toluene, cyclohexane and n-hexane., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase ( DszB ), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial biotechnology. Based on crystallographic evidence, we hypothesized that DszB undergoes an active site conformational change associated with the catalytic mechanism. Moreover, we anticipated this conformational change is responsible, in part, for enhancing product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BL21 to test these hypotheses. Activity and the resulting conformational change of DszB in the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and was inhibited via competitive binding of the product, HBP. Using circular dichroism, global changes in DszB conformation were monitored in response to HBP concentration, which indicated that both product and substrate produced similar structural changes. Molecular dynamics (MD) simulations and free energy perturbation with Hamiltonian replica exchange molecular dynamics (FEP/λ-REMD) calculations were used to investigate the molecular-level phenomena underlying the connection between conformation change and kinetic inhibition. In addition to the HBP, MD simulations of DszB bound to common, yet structurally diverse, crude oil contaminants 2',2-biphenol (BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA), and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation trajectories, including root-mean-square fluctuation (RMSF), center of mass (COM) distances, and strength of nonbonded interactions, when compared with FEP/λ-REMD calculations of ligand binding free energy, showed excellent agreement with experimentally determined inhibition constants. Together, the results show that the combination of a molecule's hydrophobicity and nonspecific interactions with nearby functional groups contributes to a competitive inhibition mechanism that locks DszB in a closed conformation and precludes substrate access to the active site.

Cyclohexanone monooxygenases (CHMOs) show very high catalytic specificity for natural Baeyer-Villiger (BV) reactions and promiscuous reduction reactions have not been reported to date. Wild-type CHMO from Acinetobacter sp. NCIMB 9871 was found to possess an innate, promiscuous ability to reduce an aromatic α-keto ester, but with poor yield and stereoselectivity. Structure-guided, site-directed mutagenesis drastically improved the catalytic carbonyl-reduction activity (yield up to 99 %) and stereoselectivity (ee up to 99 %), thereby converting this CHMO into a ketoreductase, which can reduce a range of differently substituted aromatic α-keto esters. The improved, promiscuous reduction activity of the mutant enzyme in comparison to the wild-type enzyme results from a decrease in the distance between the carbonyl moiety of the substrate and the hydrogen atom on N5 of the reduced flavin adenine dinucleotide (FAD) cofactor, as confirmed using docking and molecular dynamics simulations., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The haloalkane dehalogenase DhaA can degrade sulfur mustard (2,2'-dichlorethyl sulfide; also known by its military designation HD) in a rapid and environmentally safe manner. However, DhaA is sensitive to temperature and pH, which limits its applications in natural or harsh environments. Spore surface display technology using resistant spores as a carrier to ensure enzymatic activity can reduce production costs and extend the range of applications of DhaA. To this end, we cloned recombinant Bacillus subtilis spores pHY300PLK-cotg-dhaa-6his/DB104(FH01) for the delivery of DhaA from Rhodococcus rhodochrous NCIMB 13064. A dot blotting showed that the fusion protein CotG-linker-DhaA accounted for 0.41% ± 0.03% (P < 0.01) of total spore coat proteins. Immunofluorescence analyses confirmed that DhaA was displayed on the spore surface. The hydrolyzing activity of DhaA displayed on spores towards the HD analog 2-chloroethyl ethylsulfide was 1.74 ± 0.06 U/mL (P < 0.01), with a specific activity was 0.34 ± 0.04 U/mg (P < 0.01). This is the first demonstration that DhaA displayed on the surface of B. subtilis spores retains enzymatic activity, which suggests that it can be used effectively in real-world applications including bioremediation of contaminated environments.

Trans-o- hydroxybenzylidenepyruvate ( t HBPA) hydratase-aldolase (RnoE) catalyzes the conversion of t HBPA to 2-hydroxybenzaldehyde and pyruvate. We purified RnoE from Rhodococcus opacus and characterized its enzymatic properties. It exhibited maximum enzyme activity at 60°C and catalyzed the reverse reaction, converting various aromatic benzaldehydes and pyruvate to benzylidenepyruvate, indicating that this enzyme can be adapted for the enzymatic synthesis of α, β-unsaturated ketones.