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

Montersino, S.; Orrù, Roberto; Barendregt, Arjan; Westphal, Adrie H.; Duijn, Esther van; Mattevi, Andrea; Berkel, Willem J.H. van

doi:10.1074/jbc.m113.479303

Cited by 48 publications

(80 citation statements)

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“…S4 in the supplemental material, the enzyme activity decreased significantly with an increase in the chloride ion level, which was consistent with a previously report (9). Many flavoprotein hydroxylases have been reported to be inhibited by chloride ions, such as 3-hydroxyphenylacetate 6-hydroxylase (32) and 3-hydroxybenzoate 6-hydroxylase (33). It was generally thought that negatively charged chlorine could interfere with the binding and/or reactivity of the enzyme with oxygen, NADH, or both.…”

Section: Resultssupporting

confidence: 79%

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

Chao

Chen

Fang

et al. 2016

Appl Environ Microbiol

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cIn addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His 6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His 6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg ؊1 and showed apparent K m values of 11.40 ؎ 3.05 M for 4-hydroxyisophthalate with NADPH and 11.23 ؎ 2.43 M with NADH and similar K m values for NADPH and NADH (64.31 ؎ 13.16 and 72.76 ؎ 12.06 M, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His 6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds. The compound 2,4-xylenol (2,4-dimethylphenol), one of the six isomers of xylenol, is derived from cresylic acid or the tar acid fraction of coal tar. It is listed as a priority pollutant by the U.S. Environmental Protection Agency because of its environmental toxicity. Given the potential of 2,4-xylenol to cause harm to human health, including severe irritation of the skin and eyes and damage to the liver and kidneys, much interest has been focused on the understanding of its degradation by microorganisms. So far, several bacterial strains have been isolated for the transformation of 2,4-xylenol, such as Pseudomonas sp. (1), Pseudomonas putida NCIMB 9866 (2), Paracoccus sp. strain U120 (3), P. putida EKII (4), and Alcaligenes eutrophus JMP 134 (5, 6). Of these strains, only U120 is able to mineralize 2,4-xylenol under anaerobic conditions (3). Nevertheless, P. putida NCIMB 9866 is the only reported microorganism capable of mineralization of 2,4-xylenol under aerobic conditions (2).The early studies of 2,4-xylenol catabolism by P. putida NCIMB 9866 in the 1960s found that it was initiated by oxidation of the para-methyl group to a carboxyl group, forming 4-hydroxy-3-methylbenzoate via two putative intermediates of 4-hydroxy-3-methylbenzyl alcohol and 4-hydroxy-3-methylbenzaldehyde. The or...

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Section: Resultssupporting

confidence: 79%

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

Chao

Chen

Fang

et al. 2016

Appl Environ Microbiol

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“…Calculation of k cat according to Equation 1 at saturation concentrations of all sub- 3HB6H (para-hydroxylation). A, active site of PHBH (1PBE) (41); B, active site of 3HB6H (4BK1) (17). The crystal structure of 3HB6H was obtained from co-crystallization of 3HB and the H213S variant of 3HB6H (17).…”

Section: Discussionmentioning

confidence: 99%

“…A, active site of PHBH (1PBE) (41); B, active site of 3HB6H (4BK1) (17). The crystal structure of 3HB6H was obtained from co-crystallization of 3HB and the H213S variant of 3HB6H (17). A residue His-213 in the wild-type enzyme was modeled by replacing Ser-213 in the variant structure with His.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, 3HB6H is potentially useful for synthesizing gentisate compounds that are drugs or antioxidants (15,16). Recently, the crystal structures of the wild-type and mutant 3HB6H enzymes were solved (17). Although the overall structure of 3HB6H is similar to other single-component flavoprotein aromatic hydroxylases, the arrangement of the active site residues is quite different from ortho-hydroxylation enzymes such as PHBH.…”

Section: °C) This Intermediate Is Subsequently Protonated To Form Amentioning

confidence: 99%

“…Although the overall structure of 3HB6H is similar to other single-component flavoprotein aromatic hydroxylases, the arrangement of the active site residues is quite different from ortho-hydroxylation enzymes such as PHBH. The difference in the active site environment is presumably important for the regio-specific para-hydroxylation process (17).…”

Section: °C) This Intermediate Is Subsequently Protonated To Form Amentioning

confidence: 99%

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

Sucharitakul

Tongsook

Pakotiprapha

et al. 2013

Journal of Biological Chemistry

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Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein hydroxylase catalyzing the para-hydroxylation of 3-hydroxybenzoate. Results: The oxidative half-reaction was studied using transient kinetics. The enzyme reaction mechanism was elucidated. Conclusion:The product release and the hydroxylation limit the turnovers and the enzyme shows characteristics different from the ortho-hydroxylation enzymes. Significance: This is the first report on the oxygenation mechanism of a para-hydroxylating flavoenzyme.

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Biokatalytische Oxidationsreaktionen – aus der Sicht eines Chemikers

et al. 2018

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Enzyme gewinnen immer mehr Bedeutung als Katalysatoren für organische Oxidationsreaktionen. Mit Oxidoreduktasen stehen dem Chemiker vielseitige Werkzeuge für selektive und effiziente Oxidationsreaktionen zur Verfügung. Ihr Spektrum reicht von einfachen Alkoholoxidationen bis hin zur stereoselektiven Halogenierung nicht‐aktivierter C‐H‐Bindungen. Für viele biokatalytische Oxidationen ist bisher kein chemisches Pendant bekannt. Daher ermöglichen Oxidoreduktasen kürzere Syntheserouten. Darüber hinaus sind die generell milderen Reaktionsbedingungen biokatalytischer Oxidationen attraktiv. In diesem Aufsatz fassen wir einige aktuelle Entwicklungen im Bereich der biokatalytischen Oxidation zusammen. Wir beschreiben auch derzeitige Grenzen und vielversprechende Lösungsansätze.

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

Cited by 48 publications

References 50 publications

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866

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

Biokatalytische Oxidationsreaktionen – aus der Sicht eines Chemikers

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