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Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

ABSTRACT: Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 A resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

SUBMITTER: Dumitru R

PROVIDER: S-EPMC3109874 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

Dumitru Razvan R Jiang Wen Zhi WZ Weeks Donald P DP Wilson Mark A MA

Journal of molecular biology 20090715 2

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 A resolution. The structures show that the DMO act ...[more]

PMID: 19616011

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Project description:Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Project description:BACKGROUND:Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex. RESULTS:In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00?Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding. CONCLUSIONS:The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O-O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Dataset Information

Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

Publications

Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

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