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Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification., (Copyright © 2024. Published by Elsevier Inc.)

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins., (Copyright © 2024. Published by Elsevier Inc.)

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.)

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A., (© 2021. The Author(s).)

Allicin is a component of the characteristic smell and flavor of garlic ( Allium sativum ). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S -allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with l-cysteine, N -acetyl-l-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly ( K D of ∼2 μm) to AsFMO and that the hydride transfer occurs with pro- R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP + , reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the k cat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the k cat The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Valentino et al.)

The utilization of rubber (poly (cis-1,4-isoprene)) by rubber-degrading bacteria depends on the synthesis of rubber oxygenases that cleave the polymer extracellularly to low molecular weight products that can be taken up and used as a carbon source. All so far described Gram-negative rubber-degrading species use two related ≈ 70 kDa rubber oxygenases (RoxA and RoxB) for the primary attack of rubber while all described Gram-positive rubber-degrading strains use RoxA/RoxB-unrelated latex-clearing proteins (Lcps, ≈ 40 kDa) as rubber oxygenase(s). In this study, we identified an lcp orthologue in a Gram-negative species (Solimonas fluminis). We cloned and heterologously expressed the lcp gene of S. fluminis HR-BB, purified the corresponding Lcp protein (Lcp HR-BB ) from recombinant Escherichia coli, and biochemically characterised the Lcp HR-BB activity. Lcp HR-BB cleaved polyisoprene to a mixture of C 20 and higher oligoisoprenoids at a specific activity of 1.5 U/mg. Furthermore, spectroscopic investigation identified Lcp HR-BB as a b-haem-containing protein with an oxidised, fivefold coordinated (open) haem centre. To the best of our knowledge, this is the first report that Gram-negative bacteria can have an active rubber oxygenase of the Lcp type.

The flavin-containing monooxygenases (FMOs) play an important role in drug metabolism but they also have a high potential in industrial biotransformations. Among the hitherto characterized FMOs, there was no thermostable representative, while such biocatalyst would be valuable for FMO-based applications. Through a targeted genome mining approach, we have identified a gene encoding for a putative FMO from Nitrincola lacisaponensis, an alkaliphilic extremophile bacterium. Herein, we report the biochemical and structural characterization of this newly discovered bacterial FMO (NiFMO). NiFMO can be expressed as active and soluble enzyme at high level in Escherichia coli (90-100 mg/L of culture). NiFMO is relatively thermostable (melting temperature (T m ) of 51 °C), displays high organic solvent tolerance, and accepts a broad range of substrates. The crystal structure of NiFMO was solved at 1.8 Å resolution, which allows future structure-based enzyme engineering. Altogether, NiFMO represents an interesting newly discovered enzyme with the appropriate features to develop into an industrially applied biocatalyst.

Styrene monooxygenases are soluble two-component flavoproteins that catalyze the NADH and FAD-dependent enantioselective epoxidation of styrene to styrene oxide in the aqueous phase. These enzymes present interesting mechanistic features and potential as catalysts in biotechnological applications ranging from green chemical synthesis to bioremediation. This chapter presents approaches for the expression of the reductase (SMOB, StyB) and epoxidase (SMOA, StyA) components of SMO from pET-vectors as native or N-terminally histidine-tagged proteins in commercial strains of E. coli. The two-component structure of SMO and hydrophobic nature of styrene substrate requires some special consideration in evaluating the mechanism of this enzyme. The modular composition of the enzyme allows the flavin-reduction reaction of SMOB and styrene epoxidation reaction of SMOA to be evaluated both independently and as a composite catalytic system. The freedom to independently study the reductase and epoxidase components of SMO significantly simplifies studies of equilibrium-binding and the coupling of the free energy of ligand binding to the electrochemical potential of bound FAD. In this chapter, methods of steady-state and pre-steady-state kinetic assay, experimental approaches to equilibrium-binding reactions of flavin and substrate, and determination of the electrochemical midpoint potential of FAD bound to the reductase and epoxidase components of SMO are presented. This presentation focuses on approaches that have been successfully used in the study of the wild-type styrene monooxygenase system recovered from Pseudomonas putida (S12), but similar approaches may be effective in the characterization of related two-component enzyme systems., (© 2019 Elsevier Inc. All rights reserved.)

Flavin-N5-oxide is a new intermediate in flavoenzymology. Here we describe the identification of DszA (dibenzothiophene catabolism), RutA (uracil catabolism) and HcbA1 (hexachlorobenzene catabolism) as flavin-N5-oxide-utilizing enzymes. Mechanistic analysis of these reactions suggests a model for the identification of other examples of this catalytic motif., (© 2019 Elsevier Inc. All rights reserved.)

Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni 2+ -NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Unlabelled: Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher kcat/Km values. The efficacy and industrial scalability of one of the selected variants, CefFGOS, were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS., Importance: 7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directed-evolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Type II extradiol dioxygenase, 2'-carboxy-2,3-dihydroxybiphenyl 1,2-dioxygenase (FlnD1D2) involved in the fluorene degradation pathway of Rhodococcus sp. DFA3 was purified to homogeneity from a heterologously expressing Escherichia coli. Gel filtration chromatography and SDS-PAGE suggested that FlnD1D2 is an α4β4 heterooctamer and that the molecular masses of these subunits are 30 and 9.9 kDa, respectively. The optimum pH and temperature for enzyme activity were 8.0 and 30 °C, respectively. Assessment of metal ion effects suggested that exogenously supplied Fe(2+) increases enzyme activity 3.2-fold. FlnD1D2 catalyzed meta-cleavage of 2'-carboxy-2,3-dihydroxybiphenyl homologous compounds, but not single-ring catecholic compounds. The Km and kcat/Km values of FlnD1D2 for 2,3-dihidroxybiphenyl were 97.2 μM and 1.5 × 10(-2) μM(-1)sec(-1), and for 2,2',3-trihydroxybiphenyl, they were 168.0 μM and 0.5 × 10(-2) μM(-1)sec(-1), respectively. A phylogenetic tree of the large and small subunits of type II extradiol dioxygenases suggested that FlnD1D2 constitutes a novel subgroup among heterooligomeric type II extradiol dioxygenases.

Tetrahymena thermophila transforms exogenous cholesterol into pro-vitamin D3 (7-dehydrocholesterol) with remarkable efficiency in a one-step reaction carried out by a C-7 cholesterol desaturase. The enzyme DES7 is encoded by the gene TTHERM_00310640, identified with RNAi and gene knock-out experiments, but has not yet been heterologously expressed actively in any organism. A model derived from its amino acid sequence classified DES7p as a Rieske-type oxygenase with transmembrane localization. The protein has catalytic activity, sequence and topological similarity to DAF-36/Neverland proteins involved in the synthesis of steroid hormones in insects and nematodes. Due to their structural and functional similarity, we analyzed the expression of a codon optimized DES7 gene from Tetrahymena in the insect Sf9 cell line, identified and measured the steroid metabolites formed, and extended the actual knowledge on its localization. We found that the accumulation of 7-dehydrocholesterol could be increased 16-40-fold in Spodopterafrugiperda, depending on physiological conditions, by overexpression of T. thermophila DES7. The protein was detected in the microsomal fraction, in accordance with previous reports. Although the electron transfer chain for Des7p/DAF-36/Neverland Rieske-type oxygenases is presently unknown, we identified possible donors in the ciliate and insect genomes by bioinformatic analysis. In spite of the large evolutionary distance between S. frugiperda and T. thermophila, the results indicate that there is significant functional conservation of the electron donors, since the ciliate's sterol desaturase can function in the context of the insect electron transport system. The results achieved demonstrate that DES7 is the first gene from a ciliate, coding for a microsomal enzyme, expressed in active form in an insect cell line., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

Culture-dependent and culture-independent approaches were used for extensive retrieval of the extradiol dioxygenase (EDO) gene from the environment to investigate the relationship between the EDO genes from isolated bacteria and the metagenomic EDO genes from which they were isolated. In our previous study, we identified 91 fosmid clones showing EDO enzyme activity using a metagenomic approach. In the present study, we classified all these metagenome-derived EDOs and newly isolated 88 phenol-utilizing bacteria from the same sample and identified four EDO genes from them. Of these, two EDOs had amino acid sequences similar to those reported previously in aromatic-utilizing strains, and one EDO had a sequence almost identical to that of metagenomic EDOs identified in our previous study. Unexpectedly, one EDO showed no similarity to any class I EDOs and was categorized as class II, which has not been found in past metagenomic approaches. Quantitative polymerase chain reaction (PCR) assay indicated that the low-abundance class II EDO gene can be enriched by culturing approaches. We conclude that the combined use of the two approaches can explore the gene community more extensively than their individual use., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Gordonia polyisoprenivorans strain VH2, a potent rubber-degrading actinomycete, harbors two latex clearing proteins (Lcps), which are known to be essential for the microbial degradation of rubber. However, biochemical information on the exact role of this protein in the degradation of polyisoprene was lacking. In this study, the gene encoding Lcp1VH2 was heterologously expressed in strains of Escherichia coli, the corresponding protein was purified, and its role in rubber degradation was examined by measurement of oxygen consumption as well as by chromatographic and spectroscopic methods. It turned out that active Lcp1VH2 is a monomer and is responsible for the oxidative cleavage of poly(cis-1,4-isoprene) in synthetic as well as in natural rubber by the addition of oxygen (O2) to the cis double bonds. The resulting oligomers possess repetitive isoprene units with aldehyde (CHO-CH2-) and ketone (-CH2-CO-CH3) functional groups at the termini. Two fractions with average isoprene contents of 18 and 10, respectively, were isolated, thus indicating an endocleavage mechanism. The activity of Lcp1VH2 was determined by applying a polarographic assay. Alkenes, acyclic terpenes, or other rubber-like polymers, such as poly(cis-1,4-butadiene) or poly(trans-1,4-isoprene), are not oxidatively cleaved by Lcp1VH2. The pH and temperature optima of the enzyme are at pH 7 and 30°C, respectively. Furthermore, it was demonstrated that active Lcp1VH2 is a Cu(II)-containing oxygenase that exhibits a conserved domain of unknown function which cannot be detected in any other hitherto-characterized enzyme. The results presented here indicate that this domain might represent a new protein family of oxygenases., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxA(Xsp)) is so far the only known extracellular c-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes of Haliangium ochraceum, Myxococcus fulvus, Corallococcus coralloides, and Chondromyces apiculatus. The roxA orthologs of H. ochraceum (RoxA(Hoc)), C. coralloides BO35 (RoxA(Cco)), and M. fulvus (RoxA(Mfu)) were functionally expressed in a ΔroxA Xanthomonas sp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxA(Xsp), RoxA(Mfu), and RoxA(Cco) were purified and biochemically characterized. The optimal temperature of RoxA(Cco) and RoxA(Mfu) was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxA(Cco) and RoxA(Mfu) indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxA(Xsp). Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxA(Xsp) structure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of the Xanthomonas sp. 35Y, H. ochraceum, M. fulvus, and C. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other than Xanthomonas proves that RoxA is more common among rubber degraders than was previously assumed.

Background: The present study aimed to evaluate a hypothetical Leishmania amastigote-specific protein (LiHyp1), previously identified by an immunoproteomic approach performed in Leishmania infantum, which showed homology to the super-oxygenase gene family, attempting to select a new candidate antigen for specific serodiagnosis, as well as to compose a vaccine against VL., Methodology/principal Findings: The LiHyp1 DNA sequence was cloned; the recombinant protein (rLiHyp1) was purified and evaluated for its antigenicity and immunogenicity. The rLiHyp1 protein was recognized by antibodies from sera of asymptomatic and symptomatic animals with canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of dogs vaccinated with Leish-Tec, a Brazilian commercial vaccine; with Chagas' disease or healthy animals. In addition, the immunogenicity and protective efficacy of rLiHyp1 plus saponin was evaluated in BALB/c mice challenged subcutaneously with virulent L. infantum promastigotes. rLiHyp1 plus saponin vaccinated mice showed a high and specific production of IFN-γ, IL-12, and GM-CSF after in vitro stimulation with the recombinant protein. Immunized and infected mice, as compared to the control groups (saline and saponin), showed significant reductions in the number of parasites found in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, produced mainly by CD4 T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 response could also be observed., Conclusions/significance: The present study showed that this Leishmania oxygenase amastigote-specific protein can be used for a more sensitive and specific serodiagnosis of asymptomatic and symptomatic CVL and, when combined with a Th1-type adjuvant, can also be employ as a candidate antigen to develop vaccines against VL.

The search for novel immunogenic polypeptides to improve the accuracy and reliability of serologic diagnostic methods for Burkholderia pseudomallei infection is ongoing. We employed a rapid and efficient approach to identify such polypeptides with sera from melioidosis patients using a small insert genomic expression library created from clinically confirmed local virulent isolates of B. pseudomallei. After 2 rounds of immunoscreening, 6 sero-positive clones expressing immunogenic peptides were sequenced and their identities were: benzoate 1,2-dioxygenase beta subunit, a putative 200 kDa antigen p200, phosphotransferase enzyme family protein, short chain dehydrogenase and 2 hypothetical proteins. These immunogens were then transferred to an ELISA platform for further large scale screening. By combining shotgun expression library and ELISA assays, we identified 2 polypeptides BPSS1904 (benzoate 1,2-dioxygenase beta subunit) and BPSL3130 (hypothetical protein), which had sensitivities of 78.9% and 79.4% and specificities of 88.1% and 94.8%, respectively in ELISA test, thus suggesting that both are potential candidate antigens for the serodiagnosis of infections caused by B. pseudomallei.

A putative carotenoid oxygenase from Novosphingobium aromaticivorans was purified with a specific activity of 0.8 U/mg by His-Trap affinity chromatography. The native enzyme was estimated to be a 52 kDa monomer. Enzyme activity for β-apo-8'-carotenal was maximal at pH 8.0 and 45 °C, with a half life of 15.3 h, K(m) of 21 μM, and k(cat) of 25 l/min. The enzyme exhibited cleavage activity only for carotenoids containing one β-ionone ring and its catalytic efficiency (k(cat)/K(m)) followed the order β-apo-8'-carotenal > β-apo-4'-carotenal > γ-carotene. The enzyme converted these carotenoids to β-apo-13-carotenones by cleaving their C(13)-C(14) double bonds. The oxygen atom of β-apo-13-carotenone originated not from water but from molecular oxygen. Thus, the enzyme was an apo-carotenoid 13,14-dioxygenase.

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors to flowers, fruits and other parts of most plants of the order Caryophyllales. The formation of the structural unit of all betalains, betalamic acid from the precursor amino acid 4,5-dihydroxyphenylalanine is catalyzed by the enzyme 4,5-DOPA-extradiol-dioxygenase followed by intramolecular cyclization of the 4,5-secodopa intermediate. This paper describes the purification and the molecular and functional characterization of an active 4,5-DOPA-extradiol-dioxygenase from the best-known source of betalains-Beta vulgaris-after heterologous expression in Escherichia coli. The enzyme is a monomeric protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the production of betalamic acid, the structural, chromophoric and bioactive unit of plant pigment betalains.

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

The regiospecific oxidation of aromatic amines to aryl nitro compounds is critical to the synthesis of several natural products having pharmacological importance. The arylamine N-oxygenase (AAO) from Streptomyces thioluteus (AurF) selectively oxidizes p-aminobenzoic acid to p-nitrobenzoic acid and has been the subject of investigation for its unique chemistry and substrate preferences. Little, however, is known about the biochemistry and substrate specificities of AurF homologues, which are often associated with non-ribosomal peptide synthetases or polyketide synthases and have substrate binding pockets with substantially different amino acid compositions based on sequence alignments. An AAO homolog from Pseudomonas syringae pv. phaseolicola was expressed and purified to further explore the substrate specificity and biosynthetic utility of this enzyme class. PsAAO was most active on substituted o-aminophenols at pH 9 in buffer solutions containing 40% methanol. o-Aminophenols allow both the Pseudomonas and Streptomyces AAOs to act on para-substituted arylamines having methoxy, methyl, and nitro groups, which was previously unseen. A Hammett plot of k(cat,app) vs. σ has a ρ = -1.5, indicating substrate reactivity is dependent on the electron donating effects of substituents. The mechanistic data are consistent with an amine lone pair attacking an activated oxygen atom after formation of the hydroperoxy Fe(III/III) intermediate., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Ustilago maydis, the causative agent of corn smut disease, contains two genes encoding members of the carotenoid cleavage oxygenase family, a group of enzymes that cleave double bonds in different substrates. One of them, Cco1, was formerly identified as a β-carotene cleaving enzyme. Here we elucidate the function of the protein encoded by the second gene, termed here as Ustilago maydis Resveratrol cleavage oxygenase 1 (Um Rco1). In vitro incubations of heterologously expressed and purified UM Rco1 with different carotenoid and stilbene substrates demonstrate that it cleaves the interphenyl Cα-Cβ double bond of the phytoalexin resveratrol and its derivative piceatannol. Um Rco1 exhibits a high degree of substrate specificity, as suggested by the lack of activity on carotenoids and the other resveratrol-related compounds tested. The activity of Um Rco1 was confirmed by incubation of U. maydis rco1 deletion and over-expression strains with resveratrol. Furthermore, treatment with resveratrol resulted in striking alterations of cell morphology. However, pathogenicity assays indicated that Um rco1 is largely dispensable for biotrophic development. Our work reveals Um Rco1 as the first eukaryotic resveratrol cleavage enzyme identified so far. Moreover, Um Rco1 represents a subfamily of fungal enzymes likely involved in the degradation of stilbene compounds, as suggested by the cleavage of resveratrol by homologs from Aspergillus fumigatus, Chaetomium globosum and Botryotinia fuckeliana., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Particulate methane monooxygenase (pMMO) catalyzes methane hydroxylation to methanol at ambient temperature and pressure. pMMO from Methylosinus trichosporium OB3b is one of the two pMMOs for which the protein structure was determined by X-ray crystallography. Because purified pMMO is inherently instable in vitro, it is difficult to use for time-consuming analysis. Therefore, investigations using crude enzyme preparations of pMMO are useful in some cases. In this chapter, methods for preparing pMMO from M. trichosporium OB3b to varying degrees of purity, including bacterial cells expressing pMMO, membrane fractions containing pMMO, and highly purified pMMO, are described., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Particulate methane monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The particulate methane monooxygenase (pMMO) is a multi-copper enzyme that mediates the facile conversion of methane to methanol in methanotrophic bacteria. As a membrane-bound multi-subunit metalloprotein, the highly active protein has been difficult to isolate and purify to homogeneity for biochemical and biophysical studies. In this chapter, we describe a method to overexpress pMMO with good specific activity in high yields in the intracytoplasmic membranes of the host organism, together with two protocols to isolate and purify the enzyme from pMMO-enriched membranes without loss of the copper cofactors and enzymatic activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. PnpA (PNP 4-monooxygenase) and PnpB (para-benzoquinone reductase) were shown to be involved in the initial steps of PNP catabolism via hydroquinone. We demonstrated here that PnpA also catalyzed monooxygenation of 4-nitrocatechol (4-NC) to hydroxyquinol, probably via hydroxyquinone. It was the first time that a single-component PNP monooxygenase has been shown to catalyze this conversion. PnpG encoded by a gene located in the PNP degradation cluster was purified as a His-tagged protein and identified as a hydroxyquinol dioxygenase catalyzing a ring-cleavage reaction of hydroxyquinol. Although all the genes necessary for 4-NC metabolism seemed to be present in the PNP degradation cluster in strain WBC-3, it was unable to grow on 4-NC as a sole source of carbon, nitrogen and energy. This was apparently due to the substrate's inability to trigger the expression of genes involved in degradation. Nevertheless, strain WBC-3 could completely degrade both PNP and 4-NC when PNP was used as the inducer, demonstrating its potential in bioremediation of the environment polluted by both 4-NC and PNP.

Many bacteria use extracellular signals to coordinate group behaviours, a process referred to as quorum sensing (QS). The bacterium Pseudomonas aeruginosa utilizes a complex QS system to control expression of over 300 genes, including many involved in host colonization and disease. The Pseudomonas quinolone signal (PQS) is a component of P. aeruginosa QS, and although it contributes to virulence in some models of infection, the PQS biosynthetic pathway is not fully elucidated. Here, we show that PqsH catalyses the terminal step in PQS production, synthesizing PQS in vitro using the substrates 2-heptyl-4-quinolone (HHQ), NADH and oxygen. Structure function studies reveal that the alkyl side-chain of HHQ is critical for PqsH activity with the highest activity observed for alkyl chain lengths of 7 and 9 carbons. Due to the PqsH requirement for oxygen, PQS and PQS-controlled virulence factors are not produced by anaerobic P. aeruginosa. Interestingly, anaerobic P. aeruginosa produced PQS in the absence of de novo protein synthesis upon introduction of oxygen, indicating that oxygen is the sole limiting substrate during anaerobic growth. We propose a model in which PqsH poises anaerobic P. aeruginosa to activate PQS-controlled factors immediately upon exposure to molecular oxygen., (© 2010 Blackwell Publishing Ltd.)

Isoflavones are known to possess medicinal properties and implicated in plant-pathogen interaction. We have for the first time isolated and functionally characterized an isoflavones synthase (IFS) gene from a traditionally acclaimed medicinal plant Psoralea corylifolia abundantly growing in tropical and subtropical regions. The IFS catalyzes the exclusive reaction of phenylpropanoid pathway in leguminous plants to produce isoflavones. The full-length cDNA (PcIFS) of the gene comprised 1,563 bp and putatively encodes a polypeptide of 520 amino acid residues. The gene is expressed ubiquitously although at varying levels in different parts of the plant. The expression analysis suggests that the gene is responsive to methyl jasmonate, salicylic acid and wounding. Overexpression of PcIFS in non-leguminous tobacco plant led to the accumulation of isoflavones in petal tissue, suggesting it a functional gene from P. corylifolia involved in isoflavones biosynthesis.

Only few biological functions have been related with flavin-containing monooxygenases (FMOs) in plants, such as specific roles in auxin biosynthesis, pathogen defense, and metabolism of glucosinolates. Biochemical characterization using recombinant proteins is a promising approach to determine the precise specificity of plant FMOs for potential natural substrates. FMOs may be very difficult to express in a soluble form due to their highly hydrophobic nature and this can be improved by fusing them to solubility-enhancing proteins, such as maltose-binding protein (MBP) and N-utilization substance A (NusA). Here we describe the expression of a recombinant FMO from Coffea arabica as a maltose-binding protein fusion in Escherichia coli and its purification by affinity chromatography, producing a ready-to-use protein for enzymatic activity assays.

Biodegradation of the laundry surfactant linear alkylbenzenesulfonate (LAS) involves complex bacterial communities. The known heterotrophic community has two tiers. First, all LAS congeners are oxygenated and oxidized to about 50 sulfophenylcarboxylates (SPC). Second, the SPCs are mineralized. Comamonas testosteroni KF-1 mineralizes 3-(4-sulfophenyl)butyrate (3-C4-SPC). During growth of strain KF-1 with 3-C4-SPC, two transient intermediates were detected in the culture medium. One intermediate was identified as 4-sulfoacetophenone (SAP) (4-acetylbenzenesulfonate) by nuclear magnetic resonance (NMR). The other was 4-sulfophenol (SP). This information allowed us to postulate a degradation pathway that comprises the removal of an acetyl moiety from (derivatized) 3-C4-SPC, followed by a Baeyer-Villiger monooxygenation of SAP and subsequent ester cleavage to yield SP. Inducible NADPH-dependent SAP-oxygenase was detected in crude extracts of strain KF-1. The enzyme reaction involved transient formation of 4-sulfophenol acetate (SPAc), which was completely hydrolyzed to SP and acetate. SP was subject to NADH-dependent oxygenation in crude extract, and 4-sulfocatechol (SC) was subject to oxygenolytic ring cleavage. The first complete degradative pathway for an SPC can now be depicted with 3-C4-SPC: transport, ligation to a coenzyme A (CoA) ester, and manipulation to allow abstraction of acetyl-CoA to yield SAP, Baeyer-Villiger monooxygenation to SPAc, hydrolysis of the ester to acetate and SP, monooxygenation of SP to SC, the ortho ring-cleavage pathway with desulfonation, and sulfite oxidation.

Para-nitrophenol 4-monooxygenase (PnpA) plays an important role in bacterial degradation of para-nitrophenol by oxidative release of the nitro group from the aromatic ring to form p-benzoquinone. In order to understand the structural basis of the function of this enzyme, PnpA was cloned, expressed in Escherichia coli and purified. PnpA was crystallized by the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The PnpA crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 54.47, b = 77.56, c = 209.17 A, and diffracted to 2.24 A resolution.

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for beta-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates beta-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative beta-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of beta-carotene to yield two molecules of retinal. Analyses of beta-carotene and retinal contents in U. maydiscco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the beta-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.

The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one alpha-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in vitro, and found to stereospecifically hydroxylate the beta-position of glutamic acid. KtzO generates threo-L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering approaches.

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe-2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.

The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on V(max)/K(m) specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.

Betacyanins and anthocyanins, two main red flower pigments, never occur together in the same plant. Although the anthocyanin biosynthetic pathway has been well analyzed, the biosynthetic genes and the regulatory mechanism of the betacyanin biosynthesis are still obscure. We cloned two cDNAs of DOPA dioxygenase from Phytolacca americana, PaDOD1 and PaDOD2, that may be involved in the betalain biosynthesis. The deduced amino acid sequence of PaDOD1 and PaDOD2 showed approximately 80% homology to each other. The promoter regions of PaDOD1 and PaDOD2 were isolated by inverse PCR and analyzed using PLACE database. Some putative MYB, bHLH, and environmental stress-responsive transcription factor binding sites were detected in the PaDOD1 and PaDOD2 promoter regions. Expression patterns of PaDOD1 and PaDOD2 in suspension cultures of P. americana were investigated by semiquantitative RT-PCR. The transcripts of PaDODs were found in both betacyanin-producing red cells and non-betacyanin-producing white cells, suggesting that not only the expression of DOD, but also the supplementation of DOPA might be a regulatory step for the betalain biosynthesis in P. americana.

Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.

Background: Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO)., Results: We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191-345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively., Conclusion: The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.

The objective of this study was to enhance the activity of aniline dioxygenase (AtdA), a multi-component Rieske non-heme iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA, so as to create an enhanced biocatalyst for the bioremediation of aromatic amines. Previously, the mutation V205A was found to widen the substrate specificity of AtdA to accept 2-isopropylaniline (2IPA) for which the wild-type enzyme has no activity (Ang EL, Obbard JP, Zhao HM, FEBS J, 274:928-939, 2007). Using mutant V205A as the parent and applying one round of saturation mutagenesis followed by a round of random mutagenesis, the activity of the final mutant, 3-R21, was increased by 8.9-, 98.0-, and 2.0-fold for aniline, 2,4-dimethylaniline (24DMA), and 2-isopropylaniline (2IPA), respectively, over the mutant V205A. In particular, the activity of the mutant 3-R21 for 24DMA, which is a carcinogenic aromatic amine pollutant, was increased by 3.5-fold over the wild-type AtdA, while the AN activity was restored to the wild-type level, thus yielding a mutant aniline dioxygenase with enhanced activity and capable of hydroxylating a wider range of aromatic amines than the wild type.

Rats are a common animal model for metabolism and toxicity studies. Previously, the enzymatic properties of rat flavin-containing monooxygenase (FMO) 1 purified from hepatic and renal microsomes and that of FMO3 purified from hepatic microsomes were characterized. This study investigated the physical, immunological, and enzymatic properties of FMO3 purified from male rat kidney microsomes and compared the results with those obtained with isolated rat liver FMO3. Renal FMO3 was purified via affinity columns based on the elution of L-methionine (Met) S-oxidase activity and reactivity of the eluted proteins with human FMO3 antibody. In general, Met S-oxidase-specific activity was increased 100-fold through the purification steps. The resulting protein had similar mobility (approximately 56 kDa) as isolated rat liver FMO3 and cDNA-expressed human FMO3 by SDS-polyacrylamide gel electrophoresis. When the isolated kidney protein band was subjected to trypsin digestion and matrix-assisted laser desorption ionization/time of flight mass spectral analysis, 34% of the sequence of rat FMO3 was detected. The apparent K(m) and V(max) values for rat kidney FMO3 were determined using the known FMO substrates Met, seleno-L-methionine, S-allyl-L-cysteine (SAC), and methimazole (N-methyl-2-mercaptoimidazole). The stereoselectivity of the reactions with Met and SAC were also examined using high-performance liquid chromatography. The obtained kinetic and stereoselectivity results were similar to those we obtained in the present study, or those previously reported, for rat liver FMO3. Taken together, the results demonstrate many similar properties between rat hepatic and renal FMO3 forms and suggest that renal FMO3 may play an important role in kidney metabolism of xenobiotics containing sulfur and selenium atoms.

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoA gene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

Carotenoid oxygenases catalyse the cleavage of C-C double bonds forming apocarotenoids, a diverse group of compounds, including retinoids and the precursors of some phytohormones. Some apocarotenoids, like beta-ionone (C(13)), are ecologically important volatiles released by plants and cyanobacteria. In this work, we elucidated the activity of the Nostoccarotenoid cleavage dioxygenase (NosCCD, previously named NSC1) using synthetic and cyanobacterial substrates. NosCCD converted bicyclic and monocyclic xanthophylls, including myxoxanthophylls, glycosylated carotenoids that are essential for thylakoid and cell wall structure. The products identified revealed two different cleavage patterns. The first is observed with bicyclic xanthophylls and is identical with that of plant orthologues, while the second is novel and occurs upon cleavage of monocyclic substrates at the C9-C10 and C7'-C8' double bonds. These properties enable the enzyme to produce a plenitude of different C(10) and C(13) apocarotenoids. Expression analyses indicated a role of NosCCD in response to highlight stress. Western blot analyses of Nostoc cells revealed NosCCD as a soluble enzyme in the cytosol, which also accomodates NosCCD substrates. Incubation of the corresponding fraction with synthetic substrates revealed the activity of the native enzyme and confirmed its induction by highlight.

A novel indigo-producing oxygenase gene, designated ipoA (1,197 bp) was characterized from Rhodococcus sp. strain T104. Three indigo-negative mutations (A58V, P59L, and G251D) were obtained through random mutagenesis using an E. coli mutator strain. Subsequent saturation mutagenesis resulted in the identification of nine and three amino acid substitutions that restore activity in the A58V and P59L mutants, respectively. Activity was not restored in the G251D mutation by any other amino acids. Interestingly, activity in the A58V mutant, where a methyl group is only replaced by an isopropyl side chain, is restored by a variety of amino acids, including polar ones. A molecular modeling study suggests that the residues at positions 58, 59, and 251 of the T104 IpoA enzyme are far from the active site, indicating that the mutations must alter the overall structure of the enzyme.

The nonribosomally produced hydroxamate siderophore coelichelin from Streptomyces coelicolor contains the nonproteinogenic amino acids N(5)-hydroxyornithine and N(5)-hydroxyformylornithine that are important for iron assembly. The hydroxylation of the delta-amino group of L-ornithine is catalyzed by the flavin-dependent monooxygenase CchB. During the redox reaction nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen are consumed and flavin adenine dinucleotide (FAD) is needed as a cofactor. During this work the monooxygenase was biochemically characterized and it could be shown that the hydroxylation of l-ornithine is most likely the first step in the biosynthesis of the siderophore coelichelin.