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• Structural characterization of BtrK, a decarboxylase involved in the biosynthesis of butirosin, was performed. • BtrK has a structure similar to diaminopimelic acid decarboxylases (DAPDC), including the essential residues of the active site. • The molecular docking of a truncated version of the substrate indicates a hypothetical binding mode of the glutamate tethered to the Peptidyl Carrier Protein (PCP) BtrI. Butirosin is an aminoglycoside that has an (S)-4-amino-2-hydroxybutyrate (AHBA) moiety capable of preventing the attack of several aminoglycoside modifying enzymes. The biosynthesis and the attachment of the AHBA to the 2-deoxystreptamine (2-DOS) involve seven enzymes that use glutamate as a precursor. BtrK is a pyridoxal-5-phosphate (PLP)-dependent enzyme and performs the decarboxylation of a glutamyl moiety tethered to the peptidyl carrier protein BtrI during the AHBA biosynthetic pathway. The structure of BtrK was solved at 1.4 Å resolution and indicated a conserved folding. The PLP is covalently linked through a Schiff base to Lys49 and performs intensive hydrogen bond interactions with active site residues that are also conserved in other members of type IV PLP-dependent enzymes. Additionally, a docking simulation indicates the possible anchoring of a substrate fragment constituted of O - S -γ-Lglutamylpantetheine-4′-phosphate. The glutamyl moiety forms a number of hydrogen bonds with the putative active site residues of BtrK. The pantetheine moiety seems to perform only a few interactions and should adopt a more flexible conformation. The description of BtrK structure contributes to the understanding of the large family of PLP-dependent enzymes and also in this crucial step during the butirosin biosynthesis. [Display omitted] [ABSTRACT FROM AUTHOR]

The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene. [ABSTRACT FROM AUTHOR]

The enzyme aryl/alkenyl malonate decarboxylase (AMDase) catalyses the enantioselective decarboxylative protonation (EDP) of a range of disubstituted malonic acids to give homochiral carboxylic acids that are valuable synthetic intermediates. AMDase exhibits a number of advantages over the non-enzymatic EDP methods developed to date including higher enantioselectivity and more environmentally benign reaction conditions. In this report, AMDase and engineered variants have been used to produce a range of enantioenriched heteroaromatic α-hydroxycarboxylic acids, including pharmaceutical precursors, from readily accessible α-hydroxymalonates. The enzymatic method described here represents an improvement upon existing synthetic chemistry methods that have been used to produce similar compounds. The relationship between the structural features of these new substrates and the kinetics associated with their enzymatic decarboxylation is explored, which offers further insight into the mechanism of AMDase. [ABSTRACT FROM AUTHOR]

Methionol is an important volatile sulfur flavor compound, which can be produced via the Ehrlich pathway in Saccharomyces cerevisiae. Aminotransferase and decarboxylase are essential enzymes catalyzing methionol biosynthesis. In this work, two aminotransferase genes ARO8 and ARO9 and one decarboxylase gene ARO10 were introduced into S. cerevisiae S288c, respectively, via an expression vector. Over-expression of ARO8 resulted in higher aminotransferase activity than that of ARO9. And the cellular decarboxylase activity was remarkably increased by over-expression of ARO10. A co-expression vector carrying both ARO8 and ARO10 was further constructed to generate the recombinant strain S810. Shaking flask experiments showed that the methionol yield from S810 reached 1.27 g L-1, which was increased by 51.8 and 68.8% compared to that from the wild-type strain and the control strain harboring the empty vector. The fed-batch fermentation by strain S810 produced 3.24 g L-1 of methionol after 72 h of cultivation in a bioreactor. These results demonstrated that co-expression of ARO8 and ARO10 significantly boosted the methionol production. It is the first time that more than 3.0 g L-1 of methionol produced by genetically engineered yeast strain was reported by co-expression of the aminotransferase and decarboxylase via the Ehrlich pathway. [ABSTRACT FROM AUTHOR]

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds. [ABSTRACT FROM AUTHOR]

The ability to biosynthesize oxalic acid can provide beneficial functions to plants; however, uncontrolled or prolonged exposure to this strong organic acid results in multiple physiological problems. Such problems include a disruption of membrane integrity, mitochondrial function, metal chelation, and free radical formation. Recent work suggests that a CoA-dependent pathway of oxalate catabolism plays a critical role in regulating tissue oxalate concentrations in plants. Although this CoA-dependent pathway of oxalate catabolism is important, large gaps in our knowledge of the enzymes catalyzing each step remain. Evidence that an oxalyl-CoA decarboxylase (OXC) catalyzes the second step in this pathway, accelerating the conversion of oxalyl-CoA to formyl-CoA, has been reported. Induction studies revealed that OXC gene expression was upregulated in response to an exogenous oxalate supply. Phylogenetic analysis indicates that OXCs are conserved across plant species. Evolutionarily the plant OXCs can be separated into dicot and monocot classes. Multiple sequence alignments and molecular modeling suggest that OXCs have similar functionality with three conserved domains, the N-terminal PYR domain, the middle R domain, and the C-terminal PP domain. Further study of this CoA-dependent pathway of oxalate degradation would benefit efforts to develop new strategies to improve the nutrition quality of crops.

• Feasible and precise quantification of decarboxylases activity has been demonstrated. • Effective sequestration of CO 2 from decarboxylation successfully used Ca(OH) 2 solution. • Exploit carbonic anhydrase and CadA for in vivo CO 2 recovery for the first time. • The best strain reduced 62.9% CO 2 emission comparing to the individual CadA strain. • A novel approach to produce high-end cadaverine and recovery CO 2 simultaneously. Bio-based nylon is synthesized from putrescine, cadaverine, or 4-amino butyric acid through decarboxylases by a green process aimed toward a renewable nylon market. Thus, quantifying the activity of the key enzyme decarboxylase with precision and efficiency, is critical for the development of the system. Moreover, one molecular carbon dioxide releasing from the reaction should be considered in greenhouse gas effect. We established a new assay based on pH value decline by using different alkaline solution to absorb and capture the CO 2 released during decarboxylation. The pH change is corresponding to decarboxylase activity. On the other hand, carbonic anhydrase (CA), phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) genes involved in CO 2 respiration, were applied with lysine decarboxylase (CadA) for simultaneous production of cadaverine and recovery of CO 2. The correlation between lysine, glutamate, or arginine conversion and its pH changes in different decarboxylases was successfully determined using a numerical equation with a high regression value (R2 > 0.95). For cascade enzymatic reaction, RuBisCo was hardly co-expressed with CadA. Therefore, the strain with CadA and CA resulted in the release of the least amount of CO 2 among all the varieties, reaching the best CO 2 assimilation capability of −4.95 g-CO 2 /g-dry cell weight (DCW) and an acceptable DAP of 15.67 g/L. This illustrates a new trend of bio-mitigation of CO 2 with simultaneous production of a variety of desirable chemicals. [ABSTRACT FROM AUTHOR]

Non-oxidative decarboxylases belong to a unique enzyme family that does not require any cofactors. Here we report the characterization of a 2,3-dihydroxybenzoic acid (2,3-DHBA) decarboxylase (BDC) from Klebsiella pneumoniae and explore its application on the production of muconic acid. The enzyme properties were systematically studied, including the optimal temperature and pH, kinetic parameters, and substrate specificity. On this basis, we designed an artificial pathway for muconic acid production by connecting 2,3-DHBA biosynthesis with its degradation pathway. Over-expression of entCBA and the key enzymes in the shikimate pathway led to the production of 900 mgL-1 of 2,3-DHBA. Further, expression of the BDC coupled with catechol 1,2-dioxygenase achieved the conversion of 2,3-DHBA into muconic acid. Finally, assembly of the total pathway resulted in the de novo production of muconic acid up to 480 mgL-1. [ABSTRACT FROM AUTHOR]

Aims To contribute to the improvement of methods for the regulation and production of higher alcohols using micro-organisms, we assessed the yields achieved using 10 decarboxylase genes from three different yeast species ( Saccharomyces cerevisiae, Candida tropicalis and Pichia pastoris) by cloning them into vectors and overexpressing them in Escherichia coli hosts of different genotypes. Genes that produced the greatest yields in higher alcohol production were further assessed for the catalytic effects of the decarboxylase enzymes in the different E. coli hosts. Methods and Results A major metabolic pathway is structured via overexpressing a series of five genes, to detect the effect of decarboxylase on the production of higher alcohols. Results suggested that these genes can facilitate production of specific types of higher alcohols by diverse types of E. coli. We also showed that they play direct roles in the metabolic pathways that lead to production of higher alcohols in E. coli. The gene ARO10 from S. cerevisiae produced the highest yields for producing isobutanol and isopentanol in the host JM109. Significant differences were found in the types of higher alcohols and yields produced within the same host, for the genes PAD1, GAD1, SPE1 from S. cerevisiae. Similar results were observed for the genes ODC1 and gadB from Candida tropicalis and P. pastoris, respectively. Conclusions Investigation of these genes for identification of the key enzymatic steps or regulatory pathways involved in the Ehrlich metabolic network to produce higher alcohols is paramount for producing biofuels. The selected genes are promising targets for the development of improved production strains. Significance and Impact of the Study This is the first published assessment of the effects of decarboxylases from different yeast species that were expressed in E. coli, for the production of higher alcohols. Our results provide guidance for future studies about the use of yeast enzymes for transforming or constructing a new metabolic pathway utilizing E. coli for the production of target higher alcohols. [ABSTRACT FROM AUTHOR]

α-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o. [ABSTRACT FROM AUTHOR]

For almost 20 years, site-saturation mutagenesis ( SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In the present review, we provide examples of the growing use of SSM to study not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (Th DP)-dependent enzymes. We report that the use of SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave rise to results that were at odds with earlier kinetic and structural studies using alanine substitutions and also questioned their conclusions. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the Th DP cofactor is maintained. X-ray structures showed that Th DP stayed in the V-conformation even when the replacement residues were charged or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in Th DP-dependent enzymes such as benzoylformate decarboxylase, transketolase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase and the E1 component of the 2-oxoglutarate dehydrogenase complex. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a potent toxic element used in several industries and in the process contaminates air, soil, and water. Exposure of Saccharomyces cerevisiae to Cd increases the major phospholipids, and profound increase was observed in phosphatidylethanolamine (PE). In yeast, there are four different pathways contributing to the biosynthesis of PE, and contribution to PE pool through phosphatidylserine decarboxylase2 (psd2) is not significant in normal conditions. Upon Cd exposure, psd2Δ strain showed a significant decrease in major phospholipids including PE. When exposed to Cd, wild-type (WT) cells depicted an increase in ER stress and autophagy, whereas in psd2, ER stress was noted but autophagy process was impaired. The supplementation of ethanolamine did not overcome the Cd stress and also the autophagy process, whereas overexpression of PSD2 in psd2Δ increased the cellular tolerance, PE levels, and the autophagy process against Cd stress. From our studies, we can suggest that PSD2 of S. cerevisiae has an important role in PE synthesis and in autophagy process under Cd stress. [ABSTRACT FROM AUTHOR]

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non-complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2-amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on β-OH amino acids while avoiding activity on l-threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C-C bond forming cascades., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Enterococci were presented in all tested samples of raw cow milk (six samples) at the level 103-105 CFU/ml, fresh cheeses (five samples) at the level 102-106 CFU/g and semi-hard cheeses (five samples) at the level 103-105 CFU/g. All 33 isolated Enterococcus strains were screened for decarboxylase activity by usage differential growth medium and 20 of them possessed tyrosine decarboxylase activity. A collection of eight strains with the strongest decarboxylase activity were identified by species specific PCR as E. faecium (Z3, Z4, Br4 and 6/4D strains) and E. faecalis (Ž4, 3/3C and 4/1A strains). Enterococcus spp. Z1 strain was not identified at the species level by used methods, but the genus was confirmed by PCR method. Their tyrosin decarboxylase activity was confirmed by TLC and detection of tdc gene. Z1, Z3 and Z4 strains showed also histidine decarboxylase activity on the differential growth medium with histidine, but this activity was evaluated by TLC as a false positive reaction of medium.

Highlights: [•] Significantly enhanced conversions of ortho-carboxylation in presence of water-miscible organic co-solvents. [•] Broad operating window in terms of reaction temperature, substrate concentration and bicarbonate source. [•] Reusability of the biocatalyst over several cycles without loss of activity. [Copyright &y& Elsevier]

Background: In the anaerobic pathway of cobalamin (vitamin B12) synthesis, the CbiT enzyme plays two roles, as a cobalt-precorrin-7 C15-methyltransferase and a C12-decarboxylase, to produce the intermediate, cobalt-precorrin 8. Results: The primary structure of the hypothetical protein MJ0391, from Methanocaldococcus jannaschii, suggested that MJ0391 is a putative CbiT. Here, we report the crystal structure of MJ0391, solved by the MAD procedure and refined to final R-factor and R-free values of 19.8 & 27.3%, respectively, at 2.3 Å resolution. The asymmetric unit contains two NCS molecules, and the intact tetramer generated by crystallographic symmetry may be functionally important. The overall tertiary structure and the tetrameric arrangements are highly homologous to those found in MT0146/CbiT from Methanobacterium thermoautotrophicum. Conclusions: The conservation of functional residues in the binding site for the co-factor, AdoMet, and in the putative precorrin-7 binding pocket suggested that MJ0391 may also possess CbiT activity. The putative function of MJ0391 is discussed, based on structural homology. [ABSTRACT FROM AUTHOR]

Einen Umweg nehmen: Für die Synthese von Isovaleryl ‐ Coenzym A (CoA) in Myxobakterien wurde kürzlich ein alternativer Stoffwechselweg vorgeschlagen, der unter Leucin ‐ Mangel hochaktiv ist. Die enzymatischen Schritte dieser zuvor unbekannten Biosyntheseroute werden charakterisiert, einschließlich der Identifizierung einer bislang unbekannten 3 ‐ Methylglutaconyl ‐ CoA ‐ Decarboxylase, die wahrscheinlich aus einer CoA ‐ Transferase evolvierte. [ABSTRACT FROM AUTHOR]

Take a detour: An alternative pathway to synthesize isovaleryl coenzyme A (CoA) has recently been suggested in myxobacteria, which is highly active when leucine is limited. Each enzymatic step of this unprecedented route has now been characterized and a novel 3‐methylglutaconyl CoA decarboxylase identified that has apparently evolved from CoA transferases. [ABSTRACT FROM AUTHOR]

Abstract: In Proteus mirabilis, a putative phenylalanine decarboxylase (DisA) acts in a regulatory pathway to inhibit class 2 flagellar gene expression and motility. In this study, we demonstrate that DisA expression in Escherichia coli blocked motility and resulted in a 50-fold decrease in the expression of class 2 (fliA) and class 3 (fliC) flagellar genes. However, the expression of flhDC encoding the class 1 activator of the flagellar cascade was unchanged by DisA expression at both the transcriptional and translational levels. Phenethylamine, a decarboxylation product derived from phenylalanine, was able to mimic DisA overexpression and decrease both motility and class 2/3 flagellar gene expression. In addition, both DisA overexpression and phenethylamine strongly inhibited biofilm formation in E. coli. DisA overexpression and exogenous phenethylamine could also reduce motility in other enteric bacteria, but had no effect on motility in non-enteric Gram-negative bacteria. It is hypothesized that phenethylamine or a closely related compound formed by the DisA decarboxylation reaction inhibits the formation or activity of the FlhD4C2 complex required for activation of class 2 genes. [Copyright &y& Elsevier]

Abstract: Tautomycetin (TMC), produced by Streptomyces sp. CK4412, is an antifungal secondary metabolite with an unusual ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Recently, TMC was identified to possess additional biological functions including T cell-specific immunosuppressive and anti-cancer activities through differential inhibition of protein phosphatases, such as PP1, PP2A, and SHP2. These findings led us to isolate and characterize its entire biosynthetic and regulatory pathway gene cluster. In silico database comparisons revealed that the deduced products of two translationally coupled genes, a 666-bp tmcJ and a 1458-bp tmcK located on the 3′-terminus of the polyketide synthase gene, were found to have amino acid sequence homologies with putative bacterial decarboxylase genes. Targeted gene disruption of tmcK, but not tmcJ, from the Streptomyces sp. CK4412 chromosome resulted in production of a 5-deoxy-3″-carboxylic TMC. The tmcK mutant strain was functionally complemented using an integrative plasmid carrying tmcK and/or tmcJ–tmcK in order to restore TMC biosynthesis, a result suggesting that only TmcK is a functional TMC terminal decarboxylase. Unlike an authentic TMC, this engineered 5-deoxy-3″-carboxylic TMC analogue failed to show PP1 selectivity over PP2A, and it showed significantly reduced cytotoxicity against a human lung cancer cell line. These results imply that regio-specific modifications of TMC polyketide moiety, such as C3″-terminal carboxylation and/or C5-deketonization, could differentiate multiple biological activities in TMC produced from Streptomyces sp. CK4412. [Copyright &y& Elsevier]