3-hydroxybenzoate 6-hydroxylase Research Articles

A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum

BackgroundMicrobial production of aromatic chemicals is an attractive method for obtaining high-performance materials from biomass resources. A non-proteinogenic amino acid, 4-amino-3-hydroxybenzoic acid (4,3-AHBA), is expected to be a precursor of highly functional polybenzoxazole polymers; however, methods for its microbial production have not been reported. In this study, we attempted to produce 4,3-AHBA from glucose by introducing 3-hydroxylation of 4-aminobenzoic acid (4-ABA) into the metabolic pathway of an industrially relevant bacterium, Corynebacterium glutamicum.ResultsSix different 4-hydroxybenzoate 3-hydroxylases (PHBHs) were heterologously expressed in C. glutamicum strains, which were then screened for the production of 4,3-AHBA by culturing with glucose as a carbon source. The highest concentration of 4,3-AHBA was detected in the strain expressing PHBH from Caulobacter vibrioides (CvPHBH). A combination of site-directed mutagenesis in the active site and random mutagenesis via laccase-mediated colorimetric assay allowed us to obtain CvPHBH mutants that enhanced 4,3-AHBA productivity under deep-well plate culture conditions. The recombinant C. glutamicum strain expressing CvPHBHM106A/T294S and having an enhanced 4-ABA biosynthetic pathway produced 13.5 g/L (88 mM) 4,3-AHBA and 0.059 g/L (0.43 mM) precursor 4-ABA in fed-batch culture using a nutrient-rich medium. The culture of this strain in the chemically defined CGXII medium yielded 9.8 C-mol% of 4,3-AHBA from glucose, corresponding to 12.8% of the theoretical maximum yield (76.8 C-mol%) calculated using a genome-scale metabolic model of C. glutamicum.ConclusionsIdentification of PHBH mutants that could efficiently catalyze the 3-hydroxylation of 4-ABA in C. glutamicum allowed us to construct an artificial biosynthetic pathway capable of producing 4,3-AHBA on a gram-scale using glucose as the carbon source. These findings will contribute to a better understanding of enzyme-catalyzed regioselective hydroxylation of aromatic chemicals and to the diversification of biomass-derived precursors for high-performance materials.

Genetic redundancy of 4-hydroxybenzoate 3-hydroxylase genes ensures the catabolic safety of Pigmentiphaga sp. H8 in 3-bromo-4-hydroxybenzoate-contaminated habitats.

Genetic redundancy is prevalent in organisms and plays important roles in the evolution of biodiversity and adaptation to environmental perturbation. However, selective advantages of genetic redundancy in overcoming metabolic disturbance due to structural analogues have received little attention. Here, functional divergence of the three 4-hydroxybenzoate 3-hydroxylase (PHBH) genes (phbh1~3) was found in Pigmentiphaga sp. strain H8. The genes phbh1/phbh2 were responsible for 3-bromo-4-hydroxybenzoate (3-Br-4-HB, an anthropogenic pollutant) catabolism, whereas phbh3 was primarily responsible for 4-hydroxybenzoate (4-HB, a natural intermediate of lignin) catabolism. 3-Br-4-HB inhibited 4-HB catabolism by competitively binding PHBH3 and was toxic to strain H8 cells especially at high concentrations. The existence of phbh1/phbh2 not only enabled strain H8 to utilize 3-Br-4-HB but also ensured the catabolic safety of 4-HB. Molecular docking and site-directed mutagenesis analyses revealed that Val199 and Phe384 of PHBH1/PHBH2 were required for the hydroxylation activity towards 3-Br-4-HB. Phylogenetic analysis indicated that phbh1 and phbh2 originated from a common ancestor and evolved specifically in strain H8 to adapt to 3-Br-4-HB-contaminated habitats, whereas phbh3 evolved independently. This study deepens our understanding of selective advantages of genetic redundancy in prokaryote's metabolic robustness and reveals the factors driving the divergent evolution of redundant genes in adaptation to environmental perturbation.

Construction of a p-coumaric and ferulic acid auto-regulatory system in Pseudomonas putida KT2440 for protocatechuate production from lignin-derived aromatics

Lignin is a robust and underutilized aromatic heteropolymer on earth. The effective valorization of lignin into value-added products is an attractive research topic in lignocellulosic biorefineries. However, a low bioconversion rate, high process cost, low yield, and high toxicity of substrates hinder its further applications. In this study, an auto-regulatory system was developed and identified as an effective solution to diminish these challenging bottlenecks. First, a lignin-derived standard model aromatic p-coumaric acid (p-CA)-responsive biosensor was successfully developed through a series of rational engineering approaches in Pseudomonas putida KT2440. Furthermore, an auto-regulatory system was established, which rapidly coexpressed key rate-limiting enzymes, 4-hydroxybenzoate hydroxylase, vanillate-O-demethylase, and transporter HcnK under the biosensor element, to convert the mixture of p-CA and ferulic acid into a value-added platform chemical protocatechuate with a titer of 12.7 g/L. This study demonstrated that the constructed auto-regulatory platform is effective and economical for lignin valorization.

Identification and characterization of a chc gene cluster responsible for the aromatization pathway of cyclohexanecarboxylate degradation in Sinomonas cyclohexanicum ATCC 51369

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic degradation of benzoate, and also known to be a synthetic intermediate or the starter unit of biosynthesis of cellular constituents and secondary metabolites. Although two degradation pathways have been proposed, genetic information has been limited to the β-oxidation-like pathway. In this study, we identified a gene cluster, designated chcC1XTC2B1B2RAaAbAc, that is responsible for the CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated that the chc gene cluster is inducible by CHCA and that it consists of two transcriptional units, chcC1XTC2B1B2R and chcAaAbAc. Overexpression of the various genes in Escherichia coli, and purification of the recombinant proteins led to the functional characterization of ChcAaAbAc as subunits of a cytochrome P450 system responsible for CHCA hydroxylation; ChcB1 and ChcB2 as trans-4-hydroxyCHCA and cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was identified as a 4-oxoCHCA desaturase containing a covalently bound FAD; and ChcC2 was identified as a 4-oxocyclohexenecarboxylate desaturase. The binding constant of ChcAa for CHCA was found to be 0.37mM. Kinetic parameters established for ChcB1 indicated that it has a high catalytic efficiency towards 4-oxoCHCA compared to trans- or cis-4-hydroxyCHCA. The Km and Kcat values of ChcC1 for 4-oxoCHCA were 0.39mM and 44 s-1, respectively. Taken together with previous work on the identification of a pobA gene encoding a 4-hydroxybenzoate hydroxylase, we have now localized the remaining set of genes for the final degradation of protocatechuate before entry into the tricarboxylic acid cycle.

Natural diversity of FAD-dependent 4-hydroxybenzoate hydroxylases

4-Hydroxybenzoate 3-hydroxylase (PHBH) is the most extensively studied group A flavoprotein monooxygenase (FPMO). PHBH is almost exclusively found in prokaryotes, where its induction, usually as a consequence of lignin degradation, results in the regioselective formation of protocatechuate, one of the central intermediates in the global carbon cycle. In this contribution we introduce several less known FAD-dependent 4-hydroxybenzoate hydroxylases. Phylogenetic analysis showed that the enzymes discussed here reside in distinct clades of the group A FPMO family, indicating their separate divergence from a common ancestor. Protein homology modelling revealed that the fungal 4-hydroxybenzoate 3-hydroxylase PhhA is structurally related to phenol hydroxylase (PHHY) and 3-hydroxybenzoate 4-hydroxylase (3HB4H). 4-Hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Genome mining suggests that the 4HB1H activity is widespread in the fungal kingdom and might be responsible for the oxidative decarboxylation of vanillate, an import intermediate in lignin degradation. 4-Hydroxybenzoyl-CoA 1-hydroxylase (PhgA) catalyzes an intramolecular migration reaction (NIH shift) during the three-step conversion of 4-hydroxybenzoate to gentisate in certain Bacillus species. PhgA is phylogenetically related to 4-hydroxyphenylacetate 1-hydroxylase (4HPA1H). In summary, this paper shines light on the natural diversity of group A FPMOs that are involved in the aerobic microbial catabolism of 4-hydroxybenzoate.

The phytopathogen Xanthomonas campestris utilizes the divergently transcribed pobA/pobR locus for 4-hydroxybenzoic acid recognition and degradation to promote virulence.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the β-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.

A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site.

We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and we demonstrate its application in the high-throughput evolution of an oxygenase. A single round of selection followed by a facile growth assay enabled Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Structural modeling suggests precise reorganization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.

Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

Purification and Initial Characterization of 3-Hydroxybenzoate 6-Hydroxylase From a Halophilic Martelella Strain AD-3.

3-Hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway. We found a putative 3HB6H gene from a cluster that potentially encodes for gentisic acid degradation from a halophilic Martelella sp. strain AD-3. The corresponding protein was expressed with an N-terminal His-tag and purified by Ni2+-nitrilotriacetic acid affinity chromatography. The protein showed an overexpressed band of about 46 kDa by SDS–PAGE, and it was also proven that the enzyme contains FAD by absorption spectroscopy and HPLC analysis. The optimal activity of 3HB6H from strain AD-3 was observed in phosphate buffer (pH 8.0) at 37°C without salinity (NaCl) and metal salts. The Km values of 3-hydroxybenzoate 6-hydroxylase were determined to be 72.6 ± 10.1 μM and 104.1 ± 18.2 μM for 400 μM NADH and 3-hydroxybenzoate, respectively. Site-directed mutagenesis showed that residues 305, 306 and 308 are important for FAD binding. In addition, we found that Tyr221 and Gln305 of 3HB6H from strain AD-3 are involved in substrate binding.

PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74μM and 6.49 ± 0.17s-1, respectively, and those for NADH were 189.85 ± 55.70μM and 7.41 ± 1.39s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.

3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Contains a Phosphatidylinositol Cofactor.

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.

Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.

Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3HB, whereas Y217F has a Kd value for 3HB binding of 7.5 mm, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5-DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s(-1) ) is similar to that of wild-type enzyme (36 s(-1) ) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3HB.

Production of protocatechuic acid by Corynebacterium glutamicum expressing chorismate-pyruvate lyase from Escherichia coli.

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8mg/L of extracellular 4-HBA (36h) and 213 ± 29mg/L of extracellular PCA (80h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1mg/L of PCA from 50g/L of glucose after 72h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24h. The maximum production of PCA (1140.0 ± 11.6mg/L) was achieved when 117.0g/L of glucose was added over 96h of fed-batch fermentation.

Immobilization of 3-hydroxybenzoate 6-hydroxylase onto functionalized electrospun polycaprolactone ultrafine fibers: A novel heterogeneous catalyst

3-Hydroxybenzoate 6-hydroxylase (3HB6H), an enzyme that catalyzes regiospecific para-hydroxylation of aromatic compounds, was successfully immobilized onto electrospun polycaprolactone (PCL) ultrafine fibers (424±99nm in diameter). The fibers were fabricated from 13% w/w PCL (MW ∼8×104g/mol) dissolved in a mixed solvent of formic acid (25% v/v) and acetic acid (75% v/v) at an applied voltage of 16kV and a fiber collection distance of 12.5cm. Before being immobilized with 3HB6H, the surface of electrospun PCL fibers was functionalized by reacting with ethylenediamine activated with 5% v/v glutaraldehyde using aluminum sulfate as a catalyst. Effects of the immobilization process on pH tolerance and thermal stability of 3HB6H were investigated. Results indicated that 3HB6H immobilized onto PCL fibers could tolerate the changes in temperature and pH better than the free enzyme could. Therefore, the 3HB6H-immobilized PCL fibers are potentially useful as a heterogeneous catalyst under the conditions in which free 3HB6H could not endure.

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 × 10(6) 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 kcat 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.

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.

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.

Metabolism of 3-hydroxybenzoate and gentisate by strain Rhodococcus opacus 1CP

The ability of strain Rhodococcus opacus 1CP to utilize 3-hydroxybenzoate (3-HBA) and gentisate in concentrations up to 600 and 700 mg/L, respectively, as sole carbon and energy sources in liquid mineral media was demonstrated. Using high-performance liquid chromatography (HPLC) and thin-layer chromatography, 2,5-dihydroxybenzoate (gentisate) was identified as the key intermediate of 3-hydroxybenzoate transformation. In the cell-free extracts of the strain grown on 3-HBA or gentisate, the activities of 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, and maleylpyruvate isomerase were detected. During growth on 3-HBA, low activity of catechol 1,2-dioxygenase was detected. Based on the data obtained, the pathway of 3-HBA metabolism by strain R. opacus 1CP was proposed.