ABSTRACT
OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.
Subject(s)
Mixed Function Oxygenases , Nitrogen Oxides , Oxidation-Reduction , Nitrogen Oxides/metabolism , Nitrogen Oxides/chemistry , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Kinetics , Mutagenesis, Site-Directed , Flavins/metabolism , Flavins/chemistry , Models, Molecular , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , OxygenasesABSTRACT
CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.
Subject(s)
Aspartic Acid , Catalytic Domain , Mixed Function Oxygenases , Kinetics , Mixed Function Oxygenases/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Biocatalysis , Oxidation-Reduction , NADP/metabolism , NADP/chemistry , Mutagenesis, Site-DirectedABSTRACT
Zonocerus variegatus, or the painted grasshopper, is a food crop pest endemic in Western and Central Africa. Agricultural industries in these regions rely heavily on natural defense mechanisms to control the grasshopper population such as plant-secreted alkaloid compounds. In recent years, the Z. variegatus population has continued to rise due to acquired resistance to alkaloids. Here we focus on the kinetic characterization of a flavin-dependent monooxygenase, ZvFMO, that catalyzes the nitrogen oxidation of many of these alkaloid compounds and confers resistance to the insect. Expression and purification of ZvFMO through a traditional E. coli expression system was successful and provided a unique opportunity to characterize the catalytic properties of an FMO from insects. ZvFMO was found to catalyze oxidation reactions of tertiary nitrogen atoms and the sulfur of cysteamine. Using stopped-flow spectroscopy, we have determined the kinetic mechanism of ZvFMO. We assessed F383 for its involvement in substrate binding, which was previously proposed, and determined that this residue does not play a major role in binding substrates. Through molecular docking, we identified N304 and demonstrated that this residue plays a role in substrate binding. The role of K215 was studied and was shown that it plays a critical role in NAD(P)H binding and cofactor selectivity.
Subject(s)
Alkaloids , Grasshoppers , Animals , Mixed Function Oxygenases/chemistry , Escherichia coli , Molecular Docking Simulation , Kinetics , Organic Chemicals , Flavins , NitrogenABSTRACT
Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+ was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+.
Subject(s)
Acinetobacter baumannii , Mixed Function Oxygenases , Acinetobacter baumannii/enzymology , Anti-Bacterial Agents , Cadaverine , Flavins/metabolism , Kinetics , Mixed Function Oxygenases/chemistry , NADP/metabolism , Ornithine/chemistry , Putrescine , SiderophoresABSTRACT
N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of â¼400 µM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at â¼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.
Subject(s)
Hydrazines/metabolism , Organophosphorus Compounds/metabolism , Dinitrocresols , Flavin-Adenine Dinucleotide/metabolism , Flavins/metabolism , Hydroxylation/physiology , Kinetics , Mixed Function Oxygenases/metabolism , NADP/metabolism , Oxidation-Reduction , Succinic Acid/metabolismABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , FMN Reductase/metabolism , Flavin-Adenine Dinucleotide/metabolism , Mixed Function Oxygenases/metabolism , Streptomyces/enzymology , Azo Compounds/metabolism , Hydroxylation , Kinetics , NADPH Oxidases/metabolism , Oxygen ConsumptionABSTRACT
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 (KD of â¼2 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 kcat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the kcat 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.
Subject(s)
Garlic/enzymology , Oxygenases/metabolism , Biopolymers/metabolism , Cysteine/metabolism , Disulfides , Flavin-Adenine Dinucleotide/metabolism , Hydrogen Peroxide/metabolism , Hydroxylation , Kinetics , NADP/metabolism , Oxidation-Reduction , Oxygenases/chemistry , Oxygenases/isolation & purification , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Sulfinic Acids/metabolismABSTRACT
The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1-'C2' and N10-C1' bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.
Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/chemistry , Mixed Function Oxygenases/chemistry , Catalytic Domain , Crystallography, X-Ray , Flavin-Adenine Dinucleotide/chemistry , NADP/chemistry , Ornithine/chemistryABSTRACT
Halogenated organic compounds are extensively used in the cosmetic, pharmaceutical, and chemical industries. Several naturally occurring halogen-containing natural products are also produced, mainly by marine organisms. These compounds accumulate in the environment due to their chemical stability and lack of biological pathways for their degradation. However, a few enzymes have been identified that perform dehalogenation reactions in specific biological pathways and others have been identified to have secondary activities toward halogenated compounds. Various mechanisms for dehalogenation of I, Cl, Br, and F containing compounds have been elucidated. These have been grouped into reductive, oxidative, and hydrolytic mechanisms. Flavin-dependent enzymes have been shown to catalyze oxidative dehalogenation reactions utilizing the C4a-hydroperoxyflavin intermediate. In addition, flavoenzymes perform reductive dehalogenation, forming transient flavin semiquinones. Recently, flavin-dependent enzymes have also been shown to perform dehalogenation reactions where the reduced form of the flavin produces a covalent intermediate. Here, recent studies on the reactions of flavoenzymes in dehalogenation reactions, with a focus on covalent catalytic dehalogenation mechanisms, are described.
Subject(s)
Dinitrocresols/chemistry , Halogenation , Dinitrocresols/metabolism , Oxidation-ReductionABSTRACT
Flavin-dependent monooxygenases catalyze a wide variety of redox reactions in important biological processes and are responsible for the synthesis of highly complex natural products. Although much has been learned about FMO chemistry in the last ~80 years of research, several aspects of the reactions catalyzed by these enzymes remain unknown. In this review, we summarize recent advancements in the flavin-dependent monooxygenase field including aspects of flavin dynamics, formation and stabilization of reactive species, and the hydroxylation mechanism. Novel catalysis of flavin-dependent N-oxidases involving consecutive oxidations of amines to generate oximes or nitrones is presented and the biological relevance of the products is discussed. In addition, the activity of some FMOs have been shown to be essential for the virulence of several human pathogens. We also discuss the biomedical relevance of FMOs in antibiotic resistance and the efforts to identify inhibitors against some members of this important and growing family enzymes.
Subject(s)
Flavoproteins/chemistry , Mixed Function Oxygenases/chemistry , Animals , Bacteria/enzymology , Biocatalysis , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Flavins/chemistry , Flavins/metabolism , Flavoproteins/antagonists & inhibitors , Flavoproteins/metabolism , Humans , Hydroxylation , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , Protein Binding , Protein ConformationABSTRACT
Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs. Because the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologues (SCD1 and SCD5), while mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyses the formation of a cis-double bond between the ninth and tenth carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a di-iron centre, and cytochrome b5, which regenerates the di-iron centre. To understand better the structural basis of these characteristics of SCD function, here we crystallize and solve the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and the conformation of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal centre is coordinated by a unique spacial arrangement of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the di-iron centre.
Subject(s)
Stearoyl-CoA Desaturase/chemistry , Acyl Coenzyme A/chemistry , Acyl Coenzyme A/metabolism , Animals , Binding Sites , Crystallography, X-Ray , Cytochromes b5/chemistry , Cytochromes b5/metabolism , Electron Transport , Histidine/chemistry , Histidine/metabolism , Iron/metabolism , Mice , Models, Molecular , Oxygen/metabolism , Protein Structure, Tertiary , Static Electricity , Stearoyl-CoA Desaturase/metabolism , Structure-Activity RelationshipABSTRACT
The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-l-ornithine and NADP+. Crystal structures were determined for M101A in the resting state and complexed with NADP+. The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP+ complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP+ in preparation for a new catalytic cycle.
Subject(s)
Aspergillus fumigatus/enzymology , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Amino Acid Sequence , Aspergillus fumigatus/genetics , Crystallography, X-Ray , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavins/chemistry , Flavins/metabolism , Fungal Proteins/genetics , Kinetics , Mixed Function Oxygenases/genetics , Models, Molecular , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Siderophores/biosynthesis , Static ElectricityABSTRACT
Lysine is a precursor for desferrioxamine siderophore biosynthesis. The pathway is often initiated by lysine decarboxylases. However, little is known about those enzymes from Actinobacteria which represents a diverse class of desferrioxamine producers. In this study we focused on the genes grdesA form Gordonia rubripertincta CWB2 and psdesA from Pimelobacter simplex VkMAC-2033D that encode decarboxylases presumed to be involved in the synthesis of desferrioxamine siderophores. The corresponding proteins GrDesA and PsDesA, were heterologously produced in Escherichia coli and purified. PsDesA was isolated bound to the cofactor pyridoxal 5-phosphate and GrDesA was purified in its apo form. PsDesA showed a moderate substrate preference for lysine (Km = 0.17 mM, kcat = 0.26 s-1) compared to ornithine (Km = 0.13 mM, kcat = 0.14 s-1), while GrDesA exhibited specificity for lysine (Km = 0.13 mM, kcat = 1.2 s-1) compared to ornithine (Km = 2.9 mM, kcat = 0.18 s-1). The maximum decarboxylase activity of PsDesA was achieved at pH 7.5 at 35 °C, although PsDesA was stable up to 40°, its relative activity decreased significantly at 50 °C. The temperature optimum (40 °C) and thermostability of GrDesA were likewise, but it exhibited maximum activity at pH range 8.0-8.5, and sharply decreased outside of this range. The expression and characterization of these two decarboxylases provides insight into the biosynthetic pathway of desferrioxamines from G.rubripertincta and P. simplex and supports the functional annotation of related pathways.
Subject(s)
Actinobacteria/enzymology , Carboxy-Lyases/metabolism , Deferoxamine/metabolism , Ornithine Decarboxylase/metabolism , Siderophores/metabolism , Actinobacteria/metabolism , Biosynthetic Pathways , Substrate SpecificityABSTRACT
The enzyme UDP-galactopyranose mutase (UGM) represents a promising drug target for the treatment of infections with Trypanosoma cruzi. We have computed the Potential of Mean Force for the release of UDP-galactopyranose from UGM, using Umbrella Sampling simulations. The simulations revealed the conformational changes that both substrate and enzyme undergo during the process. It was determined that the galactopyranose portion of the substrate is highly mobile and that the opening/closing of the active site occurs in stages. Previously uncharacterized interactions with highly conserved residues were also identified. These findings provide new pieces of information that contribute to the rational design of drugs against T. cruzi.
Subject(s)
Intramolecular Transferases/chemistry , Intramolecular Transferases/metabolism , Molecular Dynamics Simulation , Trypanosoma cruzi/enzymology , Catalytic Domain , Galactose/metabolism , KineticsABSTRACT
The study of enzyme reaction mechanisms is fundamentally important to our understanding of biochemistry, cellular metabolism, and drug development. This Perspective focuses on the use of kinetic solvent viscosity effects (KSVEs) to study enzyme reactions. This technique is easily implemented and uses steady-state kinetic analyses to probe whether substrate binding is diffusion-controlled and whether product release is the rate-limiting step in the catalytic cycle. In addition, KSVEs can identify isomerization steps that are important for catalysis. The use of KSVEs in combination with other techniques, such as kinetic isotope effects, pH effects, and site-directed mutagenesis, can provide a detailed view of the mechanism of enzyme action. We present the basic theory, important experimental considerations, and potential outcomes and briefly discuss some examples from the literature. The derivation of the equations that are important for data analysis is also presented.
Subject(s)
Enzyme Assays/methods , Algorithms , Animals , Biocatalysis , Diffusion , Humans , Isomerism , Kinetics , Models, Molecular , Solvents/chemistry , Substrate Specificity , ViscosityABSTRACT
Rifampicin monooxygenase (RIFMO) decreases the potency of rifampicin (RIF) by converting it to oxidative products. Further decomposition of RIF has been observed in bacteria producing RIFMO and contributes to RIFMO-mediated drug resistance. Here we report the first crystal structure of RIFMO in complex with the hydroxylated RIF product. The 2.10 Å resolution structure reveals a breach of the ansa aliphatic chain of RIF between naphthoquinone C2 and amide N1. Our data suggest that RIFMO catalyzes the hydroxylation of RIF at the C2 atom followed by cleavage of the ansa linkage, which leads to inactivation of the antibiotic by preventing key contacts with the RNA polymerase target.
Subject(s)
Bacterial Proteins/chemistry , Drug Resistance, Bacterial , Mixed Function Oxygenases/chemistry , Nocardia/enzymology , Rifampin/chemistry , Crystallography, X-Ray , Protein DomainsABSTRACT
Galactose is an abundant monosaccharide found exclusively in mammals as galactopyranose (Gal p), the six-membered ring form of this sugar. In contrast, galactose appears in many pathogenic microorganisms as the five-membered ring form, galactofuranose (Gal f). Gal f biosynthesis begins with the conversion of UDP-Gal p to UDP-Gal f catalyzed by the flavoenzyme UDP-galactopyranose mutase (UGM). Because UGM is essential for the survival and proliferation of several pathogens, there is interest in understanding the catalytic mechanism to aid inhibitor development. Herein, we have used kinetic measurements and molecular dynamics simulations to explore the features of UGM that control the rate-limiting step (RLS). We show that UGM from the pathogenic fungus Aspergillus fumigatus also catalyzes the isomerization of UDP-arabinopyranose (UDP-Ara p), which differs from UDP-Gal p by lacking a -CH2-OH substituent at the C5 position of the hexose ring. Unexpectedly, the RLS changed from a chemical step for the natural substrate to product release with UDP-Ara p. This result implicated residues that contact the -CH2-OH of UDP-Gal p in controlling the mechanistic path. The mutation of one of these residues, Trp315, to Ala changed the RLS of the natural substrate to product release, similar to the wild-type enzyme with UDP-Ara p. Molecular dynamics simulations suggest that steric complementarity in the Michaelis complex is responsible for this distinct behavior. These results provide new insight into the UGM mechanism and, more generally, how steric factors in the enzyme active site control the free energy barriers along the reaction path.
Subject(s)
Aspergillus fumigatus/enzymology , Intramolecular Transferases/metabolism , Aspergillosis/microbiology , Aspergillus fumigatus/chemistry , Aspergillus fumigatus/metabolism , Crystallography, X-Ray , Galactose/analogs & derivatives , Galactose/metabolism , Humans , Intramolecular Transferases/chemistry , Isomerism , Kinetics , Models, Molecular , Protein Conformation , Substrate Specificity , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/metabolism , Uridine Diphosphate Galactose/metabolism , Uridine Diphosphate Sugars/metabolismABSTRACT
The redox-neutral reaction catalyzed by 2-haloacrylate hydratase (2-HAH) leads to the conversion of 2-chloroacrylate to pyruvate. Previous mechanistic studies demonstrated the formation of a flavin-iminium ion as an important intermediate in the 2-HAH catalytic cycle. Time-resolved flavin absorbance studies were performed in this study, and the data showed that the enzyme is capable of stabilizing both anionic and neutral flavin semiquinone species. The presence of a radical scavenger decreases the activity in a concentration-dependent manner. These data are consistent with the flavin iminium intermediate occurring by radical recombination.
Subject(s)
Acrylates/metabolism , Bacteria/enzymology , Flavins/metabolism , Flavoproteins/metabolism , Pyruvic Acid/metabolism , Bacteria/metabolism , Flavin-Adenine Dinucleotide/analogs & derivatives , Flavin-Adenine Dinucleotide/metabolism , NADP/metabolism , Oxidation-ReductionABSTRACT
The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox-neutral, 2-HAH was active only in the reduced state. A covalent flavin-substrate intermediate, consistent with the flavin-acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small-angle X-ray scattering was consistent with 2-HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2-HAH as a novel noncanonical flavoenzyme.
Subject(s)
Flavins/metabolism , Flavoproteins/metabolism , Biocatalysis , Escherichia coli/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavins/chemistry , Flavoproteins/genetics , Halogenation , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Kinetics , Mass Spectrometry , Scattering, Small Angle , X-Ray DiffractionABSTRACT
Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants.