Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

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.     

Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation

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.     

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10⁶ m⁻¹ s⁻¹ (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s⁻¹. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s⁻¹. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent k_cat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s⁻¹. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s⁻¹) and hydroxylation (36 ± 2 s⁻¹) partially control the overall turnover.     

Reduction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

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.     

Co-fermentation of lignocellulose-based glucose and inhibitory compounds for lipid synthesis by Rhodococcus jostii RHA1

The recalcitrant nature of lignocellulosic biomass entails pretreatment during which multiple byproducts (e.g., weak acids, furan derivatives, lignin-derived compounds) are generated. Such byproducts are generally inhibitory to fuel-producing microorganisms. In this study, lignin-derived monomers and acetate were co-fermented with glucose by Rhodococcus jostii RHA1 for lipid synthesis. The ability of R. jostii RHA1 to utilize acetate and representative lignin-derived monomers, namely p-coumaric acid, ferulic acid, 4-hydroxylic acid, and vanillic acid, were tested. The experimental results showed that R. jostii RHA1 utilized individual lignin monomers in varying degrees. The mixtures of inhibitory compounds at different levels showed higher toxicity than individual compounds, indicating synergistic effects of these monomers. When the mixture contained lower levels of glucose (5 g/L or below), adaptive-evolved (AE) R. jostii RHA1 utilized such inhibitory mixtures better for lipid synthesis. When the glucose levels were increased to 20 g/L or above, adaption evolution appeared to shorten the lag phase of co-fermentation but not necessarily enhance lipid production. This study demonstrated that R. jostii RHA1 was capable of utilizing commonly unfavorable carbon sources for lipid synthesis, which would also serve as a means to in situ detoxify inhibitory compounds.     

3-Hydroxybenzoate 6-hydroxylase from Pseudomonas aeruginosa

An inducible 3-hydroxybenzoate 6-hydroxylase has been purified to homogeneity from $\text{Pseudomonas aeruginosa}$. It contains FAD as a prosthetic group. 3-Hydroxybenzoate is quantitatively hydroxylated to give gentisate with equimolar consumptions of NADH and O₂. NADPH will substitute as an electron donor, and several aromatic analogues of 3-hydroxybenzoate stimulate reduced nucleotide oxidation by the enzyme with formation of both hydrogen peroxide and hydroxylated products. Of various analogues of 3-hydroxybenzoate, those substituted in 2,4,5 and 6-positions are competent substrates; partial uncoupling of electron flow from hydroxylation with concomitant formation of hydrogen peroxide and “gentisates” occurs. The “natural” product of the reaction, gentisate, is an effector in that it stimulates NADH oxidation with the formation of hydrogen peroxide. 3-hydroxybenzoate 6-hydroxylase thus resembles other flavoprotein hydroxylases in the general regulatory properties dictated by their aromatic substrates, pseudosubstrates or effectors.     

Molecular and Biochemical Characterization of 3-Hydroxybenzoate 6-Hydroxylase from Polaromonas naphthalenivorans CJ2

Prior research revealed that Polaromonas naphthalenivorans CJ2 carries and expresses genes encoding the gentisate metabolic pathway for naphthalene. These metabolic genes are split into two clusters, comprising nagRAaGHAbAcAdBFCQEDJI′-orf1-tnpA and nagR2-orf2I″KL (C. O. Jeon, M. Park, H. Ro, W. Park, and E. L. Madsen, Appl. Environ. Microbiol. 72:1086-1095, 2006). BLAST homology searches of sequences in GenBank indicated that the orf2 gene from the small cluster likely encoded a salicylate 5-hydroxylase, presumed to catalyze the conversion of salicylate into gentisate. Here, we report physiological and genetic evidence that orf2 does not encode salicylate 5-hydroxylase. Instead, we have found that orf2 encodes 3-hydroxybenzoate 6-hydroxylase, the enzyme which catalyzes the NADH-dependent conversion of 3-hydroxybenzoate into gentisate. Accordingly, we have renamed orf2 nagX. After expression in Escherichia coli, the NagX enzyme had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein. The enzyme was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Like other 3-hydroxybenzoate 6-hydroxylases, NagX utilized both NADH and NADPH as electron donors and exhibited a yellowish color, indicative of a bound flavin adenine dinucleotide. An engineered mutant of P. naphthalenivorans CJ2 defective in nagX failed to grow on 3-hydroxybenzoate but grew normally on naphthalene. These results indicate that the previously described small catabolic cluster in strain CJ2 may be multifunctional and is essential for the degradation of 3-hydroxybenzoate. Because nagX and an adjacent MarR-type regulatory gene are both closely related to homologues in Azoarcus species, this study raises questions about horizontal gene transfer events that contribute to operon evolution.     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.     

Expression in Escherichia coli of Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.     

Engineering levoglucosan metabolic pathway in <Emphasis Type="Italic">Rhodococcus jostii</Emphasis> RHA1 for lipid production

Oleaginous strains of Rhodococcus including R. jostii RHA1 have attracted considerable attention due to their ability to accumulate triacylglycerols (TAGs), robust growth properties and genetic tractability. In this study, a novel metabolic pathway was introduced into R. jostii by heterogenous expression of the well-characterized gene, lgk encoding levoglucosan kinase from Lipomyces starkeyi YZ-215. This enables the recombinant R. jostii RHA1 to produce TAGs from the anhydrous sugar, levoglucosan, which can be generated efficiently as the major molecule from the pyrolysis of cellulose. The recombinant R. jostii RHA1 could grow on levoglucosan as the sole carbon source, and the consumption rate of levoglucosan was determined. Furthermore, expression of one more copy of lgk increased the enzymatic activity of LGK in the recombinant. However, the growth performance of the recombinant bearing two copies of lgk on levoglucosan was not improved. Although expression of lgk in the recombinants was not repressed by the glucose present in the media, glucose in the sugar mixture still affected consumption of levoglucosan. Under nitrogen limiting conditions, lipid produced from levoglucosan by the recombinant bearing lgk was up to 43.54 % of the cell dry weight, which was comparable to the content of lipid accumulated from glucose. This work demonstrated the technical feasibility of producing lipid from levoglucosan, an anhydrosugar derived from the pyrolysis of lignocellulosic materials, by the genetically modified rhodococci strains.     

Lysine ε-aminotransferases: kinetic constants,substrate specificities,and the variation in active site residues

l-Lysine ε-aminotransferase (lysAT) is an important enzyme in tailoring the terminal amino group of l-lysine or l-ornithine and can be directed to the synthesis of various value-added chemicals such as adipic acid. Three lysATs, lysAT from Saccharopolyspora erythraea NRRL 2338 (lysAT_Sery), lysAT from Nocardia farcinica IFM 10152, and lysAT from Rhodococcus jostii RHA1, were cloned, and their kinetic values and substrate specificities were investigated. In the reaction using 5 mM l-lysine and 10 mM α-ketoglutarate, lysAT_Sery from S. erythraea NRRL 2338 showed 72% higher specific activity than lysAT from Nocardia farcinica IFM 10152 and 42% higher specific activity than lysAT from R. jostii RHA1. More interesting result was that lysAT Sery, exhibiting the highest activity among three lysATs, did not show any activity to l-ornithine. The alignment of 146 lysAT sequences from RefSeq database was searched by the EC number of lysAT to compare the active site residues among the lysAT sequences. The sequence alignment showed that only two residues, corresponding to Ala129 and Asn328 of lysAT from Mycobacterium tuberculosis H37Rv (lysAT_Mtub), showed variations among the active site residues. All the active site residues except those two residues were completely conserved throughout 145 lysAT sequences. lysAT from S. erythraea NRRL 2338 has A129T and N328S variations (residue numbers are those of the crystal structure of lysAT_Mtub). The structural analysis by the homology model indicate that Thr126 by A129T variation in lysAT_Sery is appeared to interact more tightly with the phosphate group of PLP than alanine (the distance between Thr126 and the phosphate group of PLP was 2.92 Å). In addition, Ser328 is located at the substrate recognition site of active site and, therefore, N328S variation may be connected to the substrate specificity of lysAT.     

Engineering of an L-arabinose metabolic pathway in <Emphasis Type="Italic">Rhodococcus jostii</Emphasis> RHA1 for biofuel production

The oleaginous bacterium, Rhodococcus jostii RHA1 has attracted considerable attention due to its capability to accumulate significant levels of triacylglycerol as renewable hydrocarbon. To enable the strain to utilize arabinose derived from lignocellulosic biomass, the metabolic pathway of L-arabinose utilization was introduced into R. jostii RHA1 by heterogenous expression of the operon, araBAD from Escherichia coli. The results showed that recombinant bearing araBAD could grow on L-arabinose as the sole carbon source, and additional expression of araFGH encoding the arabinose transporter from E. coli could improve the cell biomass yield from high contents of arabinose. We further increased the content of lipid produced from arabinose in the recombinants from 47.9 to 56.8 % of the cell dry weight (CDW) by overexpression of a gene, atf1 encoding a diglyceride acyltransferase from R. opacus PD630. This work demonstrated the feasibility of producing lipid from arabinose by genetic modification of the rhodococci strain.     

Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria

Comamonas testosteroni strain CNB-1 was isolated from activated sludge and has been investigated for its ability to degrade 4-chloronitrobenzene. Results from this study showed that strain CNB-1 grew on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as carbon and energy sources. Proteomic data and enzyme activity assays suggested that vanillate, 3HB, and 4HB were degraded in strain CNB-1 via protocatechuate (PCA) 4,5-cleavage pathway. The genetics and biochemistry of the PCA 4,5-cleavage pathway were investigated. Results showed that the 4-oxalomesaconate (OMA) hydratase from C. testosteroni takes only enol-OMA as substrate. A previously functionally unknown gene pmdU encodes an OMA tautomerase and catalyzes conversion of OMA_keto into OMA_enol. The 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes the last step of the PCA 4,5-cleavage pathway. We explored the 1,183 microbial genomes at GenBank for potential PCA 4,5-cleavage pathways, and 33 putative pmd clusters were found. Results suggest that PCA 4,5-cleavage pathways are mainly distributed in α- and β-Proteobacteria.     

Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

BackgoundStudying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy.MethodsWe report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (k_cat/K_m) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.ResultsAll enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.ConclusionsResults support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.General significanceResults presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Expanding the biocatalytic toolbox of flavoprotein monooxygenases from Rhodococcus jostii RHA1

With the aim to enlarge the set of available flavoprotein monooxygenases, we have cloned 8 unexplored genes from Rhodococcus jostii RHA1 that were predicted to encode class B flavoprotein monooxygenases. Each monooxygenase can be expressed as soluble protein and has been tested for conversion of sulfides and ketones. Not only enantioselective sulfoxidations, but also enantioselective Baeyer–Villiger oxidations could be performed with this set of monooxygenases. Interestingly, in contrast to known class B flavoprotein monooxygenases, all studied biocatalysts showed no nicotinamide coenzyme preference. This feature coincides with the fact that the respective sequences appear to form a discrete group of sequence related proteins, distinct from the known class B flavoprotein monooxygenases subclasses: the so-called flavin-containing monooxygenases (FMOs), N-hydroxylating monooxygenases (NMOs) and Type I Baeyer–Villiger monooxygenases (BVMOs). Taken together, these data reveal the existence of a new subclass of class B flavoprotein monooxygenases, which we coined as Type II FMOs, that can perform Baeyer–Villiger oxidations and accept both NADPH and NADH as coenzyme. The uncovered biocatalytic properties of the studied Type II FMOs make this newly recognized subclass of monooxygenases of potential interest for biocatalytic applications.