Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

Yu, Yimin; Wang, Lee Ho; Tu, Shiao‐Chun

doi:10.1021/bi00378a018

Cited by 15 publications

(7 citation statements)

References 27 publications

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“…However, 3-hydroxybenzoate 6-hydroxylase was found to retain 57% of maximum activity at 10°C, which is consistent with strain CJ2's relatively low optimum growth temperature of 20°C (11). The effect of FAD on the activity of the purified NagX protein was investigated because the recombinant NagX protein showed a yellowish color, indicative of a bound flavin, and FAD has been shown to be a prosthetic group in other 3-hydroxybenzoate 6-hydroxylases (8,23,30,33,35). The addition of 10 M FAD increased the activity of the purified recombinant 6-hydroxylase approximately fourfold.…”

Section: Vol 73 2007supporting

confidence: 57%

“…The NagX enzyme from strain CJ2 was able to convert 3-hydroxybenzoate into gentisate without salicylate 5-hydroxylase activity. Active recombinant NagX had an approximate molecular mass of 43 kDa, as estimated by gel filtration, and was probably a monomeric protein, similar to the 3-hydroxybenzoate 6-hydroxylases from P. cepacia (44 kDa) (33,35) and K. pneumoniae M5a1 (42 kDa) (30). Like other characterized 3-hydroxybenzoate 6-hydroxylase enzymes, NagX utilizes NADH as well as NADPH as the electron donor for the reduction of the flavin cofactor, FAD.…”

Section: Discussionmentioning

confidence: 89%

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

Park¹,

Jeon²,

Jang³

et al. 2007

Appl Environ Microbiol

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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.Polaromonas naphthalenivorans CJ2, previously found to be responsible for the degradation of naphthalene in situ at a coal tar waste-contaminated site (10), is able to utilize naphthalene as the sole carbon source. Recent sequencing and analysis of the entire naphthalene degradation pathway of P. naphthalenivorans CJ2 (12) revealed that the naphthalene-catabolic (nag) genes in P. naphthalenivorans CJ2 are homologous to those in Ralstonia sp. strain U2, which metabolizes naphthalene via the gentisate pathway, converting naphthalene into fumarate and pyruvate via salicylate (2-hydroxybenzoate) and gentisate (2,5-dihydroxybenzoate) (5). This finding has recently been confirmed biochemically (G. M. Pumphrey and E. L. Madsen, submitted for publication). In contrast to the nag genes of strain U2, which are organized in a single operon, the naphthalenecatabolic genes of strain CJ2 are organized into two gene clusters (Fig. 1A), comprising nagRAaGHAbAcAdBFCQEDJIЈ-orf1-tnpA (large cluster) and nagR2-orf2IЉKL (small cluster). LysR-type (nagR) and MarR-type (nagR2) transcriptional regulators act, respectively, to up-regulate and down-regulate the growth of strain CJ2 on naphthalene in min...

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Section: Vol 73 2007supporting

confidence: 57%

Section: Discussionmentioning

confidence: 89%

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

Park¹,

Jeon²,

Jang³

et al. 2007

Appl Environ Microbiol

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“…In similar titrations with anthranilate hydroxylase (Einarsdottir et al, 1989) and melilotate hydroxylase (Strickland & Massey, 1973a) no flavin semiquinone accumulates either in the absence or in the presence of substrates. 3-Hydroxybenzoate-6-hydroxylase stabilizes a neutral flavin semiquinone either in the absence or in tje presence of m-hydroxybenzoate (Yu et al, 1987). In contrast to aromatic flavoprotein hydroxylases such as p-hydroxybenzoate hydroxylase (Howell et al, 1972), melilotate hydroxylase (Strickland & Massey, 1973b;Schopfer & Massey, 1979), and phenol hydroxylase (Detmer & Massey, 1984), which form charge-transfer complexes between FAD enzyme and pyridine nucleotides, no such species were detected during the reduction of MHPCO.…”

Section: Discussionmentioning

confidence: 99%

Thermodynamics and Reduction Kinetics Properties of 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase

et al. 1997

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The investigation by absorbance and fluorescence rapid reaction spectrophotometry of the binding of the substrate MHPC (2-methyl-3-hydroxypyridine-5-carboxylic acid) or the substrate analog 5HN (5-hydroxynicotinic acid) to the flavoprotein MHPCO (2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase) shows that the binding proceeds in two steps. An enzyme-substrate complex initially formed is followed by a ligand-induced isomerization. This binding process is required for efficient reduction of the enzyme-bound flavin, as evidenced by the fact that MHPCO-substrate complexes can be reduced by NADH much faster than the enzyme alone. Since redox potential values of MHPCO and MHPCO-substrate complexes are the same, steric factors, such as the relative orientation of MHPC to the enzyme-bound flavin, are important for efficient hydride transfer to occur.

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“…[37]. The enzyme for the conversion of 3-CBA to the 3-hydroxybenzoic acid is not yet known, the second reaction is catalyzed by 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) [49]. Monooxygenase reaction was reported in strains Pseudomonas sp.…”

Section: Degradation Of 2-chlorobenzoic Acidmentioning

confidence: 99%

Bioremediation of Chlorobenzoic Acids

Vrchotová¹,

Macková²,

Macek³

et al. 2013

Applied Bioremediation - Active and Passive Approaches

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Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

Cited by 15 publications

References 27 publications

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

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

Thermodynamics and Reduction Kinetics Properties of 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase

Bioremediation of Chlorobenzoic Acids

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