Two-component Flavin-dependent Monooxygenase Research Articles

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

A novel pbd gene cluster responsible for pyrrole and pyridine ring cleavage in Rhodococcus ruber A5

Pyridine and pyrrole, which are regarded as recalcitrant chemicals, are released into the environment as a result of industrial manufacturing processes, posing serious hazards to both the environment and human health. However, the pyrrole degradation mechanism and the pyridine-degrading gene in Rhodococcus are unknown. Herein, a highly efficient pyridine and pyrrole degradation strain Rhodococcus ruber A5 was isolated. Strain A5 completely degraded 1000 mg/L pyridine in a mineral salt medium within 24 h. The pyridine degradation of strain A5 was optimized using the BoxBehnken design. The optimum degradation conditions were found to be pH 7.15, temperature 28.06 ℃, and inoculation amount 1290.94 mg/L. The pbd gene clusters involved in pyridine degradation were discovered via proteomic analysis. The initial ring cleavage of pyridine and pyrrole in strain A5 was carried out by the two-component flavin-dependent monooxygenase PbdA/PbdE. The degradation pathways of pyridine and pyrrole were proposed by the identification of metabolites and comparisons of homologous genes. Additionally, homologous pbd gene clusters were found to exist in different bacterial genomes. Our study revealed the ring cleavage mechanisms of pyrrole and pyridine, and strain A5 was identified as a promising resource for pyridine bioremediation.

Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations.

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, β endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with β-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.

Crystal structure of MAB_4123, a putative flavin-dependent monooxygenase from Mycobacterium abscessus.

Numerous bacteria from different phylae can perform desulfurization reactions of organosulfur compounds. In these degradation or detoxification pathways, two-component flavin-dependent monooxygenases that use flavins (FMN or FAD) as a cofactor play important roles as they catalyse the first steps of these metabolic routes. The TdsC or DszC and MsuC proteins belong to this class of enzymes as they process dibenzothiophene (DBT) and methanesulfinate. Elucidation of their X-ray structures in apo, ligand-bound and cofactor-bound forms has provided important molecular insights into their catalytic reaction. Mycobacterial species have also been shown to possess a DBT degradation pathway, but no structural information is available on these two-component flavin-dependent monooxygenases. In this study, the crystal structure of the uncharacterized MAB_4123 protein from the human pathogen Mycobacterium abscessus is presented. The structure solved at high resolution displays high similarity to homologs from Rhodococcus, Paenibacillus and Pseudomonas species. In silico docking approaches suggest that MAB_4123 binds FMN and may use it as a cofactor. Structural analysis strongly suggests that MAB_4123 is a two-component flavin-dependent monooxygenase that could act as a detoxifying enzyme of organosulfur compounds in mycobacteria.

A Novel Microbial Consortia Catalysis Strategy for the Production of Hydroxytyrosol from Tyrosine.

Hydroxytyrosol, a valuable plant-derived phenolic compound, is increasingly produced from microbial fermentation. However, the promiscuity of the key enzyme HpaBC, the two-component flavin-dependent monooxygenase from Escherichia coli, often leads to low yields. To address this limitation, we developed a novel strategy utilizing microbial consortia catalysis for hydroxytyrosol production. We designed a biosynthetic pathway using tyrosine as the substrate and selected enzymes and overexpressing glutamate dehydrogenase GdhA to realize the cofactor cycling by coupling reactions catalyzed by the transaminase and the reductase. Additionally, the biosynthetic pathway was divided into two parts and performed by separate E. coli strains. Furthermore, we optimized the inoculation time, strain ratio, and pH to maximize the hydroxytyrosol yield. Glycerol and ascorbic acid were added to the co-culture, resulting in a 92% increase in hydroxytyrosol yield. Using this approach, the production of 9.2 mM hydroxytyrosol was achieved from 10 mM tyrosine. This study presents a practical approach for the microbial production of hydroxytyrosol that can be promoted to produce other value-added compounds.

Characterization of the flavin dependent dimethylsulfone monooxygenase SfnG from Pseudomonas fluorescens

Sulfur is one of the essential elements of life, being involved in pathways to generate sulfide, which is used downstream for amino acid, metabolite, and signaling molecule synthesis. Inorganic sulfate and sulfite are the typical sources of sulfur for these bacteria; however, limiting sulfate/sulfite conditions require bacteria to use alternative sulfur sources. Within Pseudomonas fluorescens a set of genes are expressed that allow them to utilize alkanesulfonates to generate sulfite in this limiting condition; the proteins expressed are two‐component flavin‐dependent monooxygenases (TC‐FMOs) involved in the desulfonation of the organosulfur compound and a flavin mononucleotide (FMN) reductase essential for the generation of reduced FMN. The first step in this pathway is SfnG, a TC‐FMO that acts upon dimethylsulfone to produce methanesulfinate, which is further acted on by enzymes in this pathway to generate sulfite. Current work has structurally characterized SfnG through X‐ray crystallography with the ligands FMN and DMSO2 to 1.75‐Å resolution and without ligands to 2.6‐Å resolution. The characterization of SfnG elucidated residues predicted to be important for binding of DMSO2 and FMN within the active site. Further structural analysis showed the active site of SfnG is enclosed by a lid region which is disordered without substrates bound, and the structural arrangement of ligands and the predicted oxygen binding site is in support of SfnG utilizing the recently proposed N5‐peroxyflavin intermediate. Further analysis of SfnG has revealed an interesting quaternary state reliant on substrate binding. The quaternary structure organization is different from other TC‐FMO’s involved in C–S bond cleavage, which was further explored through FMN binding studies. These results will contribute towards our knowledge of TC‐FMOs involved in C–S bond cleavage.

One-Pot Bioconversion of Lignin-Derived Substrates into Gallic Acid.

Lignin is regarded as the most abundant renewable aromatic compound on earth. In this study, we established Escherichia coli-based whole-cell biocatalytic systems to efficiently convert two lignin-derived substrates (ferulic acid and p-coumaric acid) to gallic acid. For the synthesis of gallic acid from ferulic acid, we used the recombinant E. coli expressing feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase from Pseudomonas putida, aldehyde dehydrogenase (HFD1) from Saccharomyces cerevisiae, vanillic acid O-demethylase (VanAB) from P. putida, and a mutant version of p-hydroxybenzoate hydroxylase (PobAY385F) from P. putida. Under the fed-batch mode, 19.57 mM gallic acid was obtained from 20 mM ferulic acid with a conversion rate of 97.9%. To achieve gallic acid synthesis from p-coumaric acid, we replaced VanAB with the two-component flavin-dependent monooxygenase (HpaBC) from E. coli. Under optimal conditions, 20 mM p-coumaric acid afforded the production of 19.96 mM gallic acid with near 100% conversion. To the best of our knowledge, our work represented the first study to develop E. coli-based whole-cell biocatalysts for the eco-friendly synthesis of gallic acid from lignin-derived renewable feedstocks.

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C–S bond cleavage, substrate scope, and an unexpected role for the tetramer

Bacterial two-component flavin-dependent monooxygenases cleave the stable C–S bond of environmental and anthropogenic organosulfur compounds. The monooxygenase MsuD converts methanesulfonate (MS−) to sulfite, completing the sulfur assimilation process during sulfate starvation, but the mechanism of this conversion remains unclear. To explore the mechanism of C–S bond cleavage, we report a series of crystal structures of MsuD from Pseudomonas fluorescens in different liganded states. This report provides the first crystal structures of an alkanesulfonate monooxygenase with a bound flavin and alkanesulfonate, elucidating the roles of the active site lid, the protein C terminus, and an active site loop in flavin and/or alkanesulfonate binding. These structures position MS− closest to the flavin N5 position, consistent with an N5-(hydro)peroxyflavin mechanism rather than a classical C4a-(hydro)peroxyflavin mechanism. A fully enclosed active site is observed in the ternary complex, mediated by interchain interaction of the C terminus at the tetramer interface. These structures identify an unexpected function of the protein C terminus in this protein family in stabilizing tetramer formation and the alkanesulfonate-binding site. Spurred by interest from the crystal structures, we conducted biochemical assays and molecular docking that redefine MsuD as a small- to medium-chain alkanesulfonate monooxygenase. Functional mutations verify the sulfonate-binding site and reveal the critical importance of the protein C terminus for monooxygenase function. These findings reveal a deeper understanding of MsuD’s functionality at the molecular level and consequently how it operates within its role as part of the sulfur assimilation pathway.

Ligand bound structure of the alkanesulfonate monooxygenase MsuD provides insight into catalysis

The production of sulfite is essential for an abundance of organisms, including many bacteria. During sulfate starvation of certain bacteria, the assimilation and conversion of sulfur from organosulfur compounds is done through usage of two-component flavin-dependent monooxygenases to cleave stable C–S bonds. The monooxygenase MsuD converts methanesulfonate to sulfite, however its mechanism is as yet unresolved. In order to elucidate the alkanesulfonate monooxygenase mechanism, the crystal structure of MsuD from Pseudomonas fluorescens was obtained with flavin and methanesulfonate bound. As a result, the first structure of an alkanesulfonate monooxygenase with bound flavin and alkanesulfonate reveal the structural changes necessary for ligand binding to position flavin and methanesulfonate for catalysis. An intricate network of interactions is observed between the protein, flavin, and methanesulfonate binding sites. Furthermore, the ternary MsuD structure has enabled us to revisit and update the C–S cleavage mechanism of two-component flavin-dependent alkanesulfonate monooxygenases, and biochemical assays spurred by these discoveries have allowed us to redefine MsuD as a small to medium chain alkanesulfonate monooxygenase.

Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis.

The flavin reductase (FRED) and isobutylamine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED:IBAH system. The affinity of FRED for FADox is higher than its affinity for FADred, consistent with its function as a flavin reductase. Conversely, IBAH binds FADred more tightly than FADox, consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 μM. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.

Developing a highly efficient hydroxytyrosol whole-cell catalyst by de-bottlenecking rate-limiting steps

Hydroxytyrosol is an antioxidant free radical scavenger that is biosynthesized from tyrosine. In metabolic engineering efforts, the use of the mouse tyrosine hydroxylase limits its production. Here, we design an efficient whole-cell catalyst of hydroxytyrosol in Escherichia coli by de-bottlenecking two rate-limiting enzymatic steps. First, we replace the mouse tyrosine hydroxylase by an engineered two-component flavin-dependent monooxygenase HpaBC of E. coli through structure-guided modeling and directed evolution. Next, we elucidate the structure of the Corynebacterium glutamicum VanR regulatory protein complexed with its inducer vanillic acid. By switching its induction specificity from vanillic acid to hydroxytyrosol, VanR is engineered into a hydroxytyrosol biosensor. Then, with this biosensor, we use in vivo-directed evolution to optimize the activity of tyramine oxidase (TYO), the second rate-limiting enzyme in hydroxytyrosol biosynthesis. The final strain reaches a 95% conversion rate of tyrosine. This study demonstrates the effectiveness of sequentially de-bottlenecking rate-limiting steps for whole-cell catalyst development.

Phenolic hydroxylases.

Many flavin-dependent phenolic hydroxylases (monooxygenases) have been extensively investigated. Their crystal structures and reaction mechanisms are well understood. These enzymes belong to groups A and D of the flavin-dependent monooxygenases and can be classified as single-component and two-component flavin-dependent monooxygenases. The insertion of molecular oxygen into the substrates catalyzed by these enzymes is beneficial for modifying the biological properties of phenolic compounds and their derivatives. This chapter provides an in-depth discussion of the structural features of single-component and two-component flavin-dependent phenolic hydroxylases. The reaction mechanisms of selected enzymes, including 3-hydroxy-benzoate 4-hydroxylase (PHBH) and 3-hydroxy-benzoate 6-hydroxylase as representatives of single-component enzymes and 3-hydroxyphenylacetate 4-hydroxylase (HPAH) as a representative of two-component enzymes, are discussed in detail. This chapter comprises the following four main parts: general reaction, structures, reaction mechanisms, and enzyme engineering for biocatalytic applications. Enzymes belonging to the same group catalyze similar reactions but have different unique structural features to control their reactivity to substrates and the formation and stabilization of C4a-hydroperoxyflavin. Protein engineering has been employed to improve the ability to use these enzymes to synthesize valuable compounds. A thorough understanding of the structural and mechanistic features controlling enzyme reactivity is useful for enzyme redesign and enzyme engineering for future biocatalytic applications.

Structure and function of the two-component flavin-dependent methanesulfinate monooxygenase within bacterial sulfur assimilation

Methyl sulfur compounds are a rich source of environmental sulfur for microorganisms, but their use requires redox systems. The bacterial sfn and msu operons contain two-component flavin-dependent monooxygenases for dimethylsulfone (DMSO2) assimilation: SfnG converts DMSO2 to methanesulfinate (MSI–), and MsuD converts methanesulfonate (MS–) to sulfite. However, the enzymatic oxidation of MSI– to MS– has not been demonstrated, and the function of the last enzyme of the msu operon (MsuC) is unresolved. We employed crystallographic and biochemical studies to identify the function of MsuC from Pseudomonas fluorescens. The crystal structure of MsuC adopts the acyl-CoA dehydrogenase fold with putative binding sites for flavin and MSI–, and functional assays of MsuC in the presence of its oxidoreductase MsuE, FMN, and NADH confirm the enzymatic generation of MS–. These studies reveal that MsuC converts MSI– to MS– in sulfite biosynthesis from DMSO2.

A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria

Sulfonamide-degrading bacteria have been discovered in various environments, suggesting the presence of novel resistance mechanisms via drug inactivation. In this study, Microbacterium sp. CJ77 capable of utilizing various sulfonamides as a sole carbon source was isolated from a composting facility. Genome and proteome analyses revealed that a gene cluster containing a flavin-dependent monooxygenase and a flavin reductase was highly up-regulated in response to sulfonamides. Biochemical analysis showed that the two-component monooxygenase system was key enzymes for the initial cleavage of sulfonamides. Co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug-inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that the gene cluster containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR) was highly conserved in a genomic island shared among sulfonamide-degrading actinobacteria, all of which also contained sul1-carrying class 1 integrons. These results suggest that the sulfonamide metabolism may have evolved in sulfonamide-resistant bacteria which had already acquired the class 1 integron under sulfonamide selection pressures. Furthermore, the presence of multiple insertion sequence elements and putative composite transposon structures containing the sulX gene cluster indicated potential mobilization. This is the first study to report that sulX responsible for both sulfonamide degradation and resistance is prevalent in sulfonamide-degrading actinobacteria and its genetic signatures indicate horizontal gene transfer of the novel resistance gene.

Investigations of two-component flavin-dependent monooxygenase systems.

Bacterial two-component flavin-dependent monooxygenase systems catalyze the oxidation of diverse metabolic reactions. There are several shared mechanistic features in the two-component monooxygenase systems that differ from canonical monooxygenase enzymes. The flavin reductases catalyze the reductive half-reaction, and the reduced flavin is transferred to the monooxygenase enzyme. The oxidative half-reaction catalyzed by the monooxygenase enzyme has been proposed to occur through the formation of a (hydro)peroxyflavin intermediate. In some two-component flavin-dependent systems the mechanism of flavin transfer involves protein-protein interactions between the flavin reductase and monooxygenase enzyme. Methods are presented that provide an alternative approach from flavin-bound monooxygenases to evaluate the kinetic properties and flavin transfer mechanism of the two-component flavin-dependent monooxygenase systems.

Two-Component Flavin-Dependent Riboflavin Monooxygenase Degrades Riboflavin in Devosia riboflavina.

The actinobacterium Microbacterium maritypicum splits riboflavin (vitamin B2) into lumichrome and d-ribose. However, such degradation by other bacteria and the involvement of a two-component flavin-dependent monooxygenase (FMO) in the reaction remain unknown. Here we investigated the mechanism of riboflavin degradation by the riboflavin-assimilating alphaproteobacterium Devosia riboflavina (formerly Pseudomonas riboflavina). We found that adding riboflavin to bacterial cultures induced riboflavin-degrading activity and a protein of the FMO family that had 67% amino acid identity with the predicted riboflavin hydrolase (RcaE) of M. maritypicum MF109. The D. riboflavina genome clustered genes encoding the predicted FMO, flavin reductase (FR), ribokinase, and flavokinase, and riboflavin induced their expression. This finding suggests that these genes constitute a mechanism for utilizing riboflavin as a carbon source. Recombinant FMO (rFMO) protein of D. riboflavina oxidized riboflavin in the presence of reduced flavin mononucleotide (FMN) provided by recombinant FR (rFR), oxidized FMN and NADH, and produced stoichiometric amounts of lumichrome and d-ribose. Further investigation of the enzymatic properties of D. riboflavina rFMO indicated that rFMO-rFR coupling accompanied O2 consumption and the generation of enzyme-bound hydroperoxy-FMN, which are characteristic of two-component FMOs. These results suggest that D. riboflavina FMO is involved in hydroperoxy-FMN-dependent mechanisms to oxygenize riboflavin and a riboflavin monooxygenase is necessary for the initial step of riboflavin degradation.IMPORTANCE Whether bacteria utilize either a monooxygenase or a hydrolase for riboflavin degradation has remained obscure. The present study found that a novel riboflavin monooxygenase, not riboflavin hydrolase, facilitated this process in D. riboflavina The riboflavin monooxygenase gene was clustered with flavin reductase, flavokinase, and ribokinase genes, and riboflavin induced their expression and riboflavin-degrading activity. The gene cluster is uniquely distributed in Devosia species and actinobacteria, which have exploited an environmental niche by developing adaptive mechanisms for riboflavin utilization.

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate-FMN-enzyme complex remains unknown. Herein, we report the crystal structures of the DBT-FMN-TdsC and DBTO-FMN-TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

The Two-Component Monooxygenase MeaXY Initiates the Downstream Pathway of Chloroacetanilide Herbicide Catabolism in Sphingomonads.

Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads.IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the para-hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative.

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

The oxygenase component (C2) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C2 with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (kOH) for 4-APA of 0.028 s-1 compared to 17 s-1 for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s-1 for 4-HPA versus 2.5 s-1 for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C2 reactivity toward a phenolic compound. Removing this hydroxyl group expands C2 activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.