ABSTRACT
3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli's cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.
ABSTRACT
Flavin-dependent monooxygenases catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. As such, they are involved in key biological processes ranging from catabolism, detoxification and biosynthesis, to light emission and axon guidance. Based on fold and function, flavin-dependent monooxygenases can be distributed into eight groups. Groups A and B comprise enzymes that rely on NAD(P)H as external electron donor. Groups C-F are two-protein systems, composed of a monooxygenase and a flavin reductase. Groups G and H comprise internal monooxygenases that reduce the flavin cofactor through substrate oxidation. Recently, many new flavin-dependent monooxygenases have been discovered. In addition to posing basic enzymological questions, these proteins attract attention of pharmaceutical and fine-chemical industries, given their importance as regio- and enantioselective biocatalysts. In this review we present an update of the classification of flavin-dependent monooxygenases and summarize the latest advances in our understanding of their catalytic and structural properties.
Subject(s)
Flavins/metabolism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Animals , Humans , Models, Molecular , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Protein ConformationABSTRACT
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.
Subject(s)
Bacterial Proteins/chemistry , Flavoproteins/chemistry , Mixed Function Oxygenases/chemistry , Phospholipids/chemistry , Rhodococcus/enzymology , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Flavoproteins/genetics , Flavoproteins/metabolism , Gene Expression , Gentisates/chemistry , Gentisates/metabolism , Hydroxylation , Mass Spectrometry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Mutation, Missense , NAD/chemistry , NAD/genetics , NAD/metabolism , Phospholipids/genetics , Phospholipids/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/geneticsABSTRACT
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M(-1) s(-1). In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s(-1). At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ~51 s(-1), whereas without 3-HB, the rate constant is 0.43 s(-1) at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme-product (2,5-DHB) complex, with a rate constant of 45 ± 2 s(-1). The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em 0 for the enzyme-substrate complex is -179 ± 1 mV compared to an E(m)(0) of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme-ligand complex to a fully activated form before flavin reduction takes place.
Subject(s)
Mixed Function Oxygenases/metabolism , Rhodococcus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Kinetics , Ligands , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , NAD/metabolism , Oxidation-Reduction , Rhodococcus/genetics , Substrate Specificity , ThermodynamicsABSTRACT
The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme.
