NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase

Hartmann, Steffen; Hultschig, Claus; Eisenreich, Wolfgang; Fuchs, Georg; Bacher, Adelbert; Ghisla, Sandro

doi:10.1073/pnas.96.14.7831

Cited by 28 publications

(27 citation statements)

References 22 publications

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“…The likely mechanism of this reaction proceeds via an arene oxide or a ketone intermediate generated by an NIH shift step involving chlorine (Scheme 1). Such a shift has been observed recently in the flavin monooxygenase-mediated oxidation of benzene compounds (42). Reduction by two electrons, loss of chloride, and protonation gives the PCP product.…”

Section: Structure-based Cyp101 Engineering For Pecb Oxidationmentioning

confidence: 92%

Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene

Chen¹,

Christopher²,

Jones³

et al. 2002

Journal of Biological Chemistry

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We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J. P., O'Hare, E. J., and Wong, L. L. (2001) Eur. J. Biochem. 268, 1460 -1467). In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene. The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction. In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron. The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines. The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101) ؊1 min ؊1 , 45 times faster than the F87W/Y96F/V247L parent mutant. The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min ؊1 . Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms. In principle, the F87W/ Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

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Section: Structure-based Cyp101 Engineering For Pecb Oxidationmentioning

confidence: 92%

Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene

Chen¹,

Christopher²,

Jones³

et al. 2002

Journal of Biological Chemistry

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“…The reduction of the flavin could be achieved by NADPH, as found in flavin-containing monooxygenases acting on anthranilate, phenol, and other aromatic substrates (14,31,35), or by reduced Fe-S centers in the protein, as in cytochrome P450 reductase, sulfite reductase, and nitrite reductase (35). Recently, a flavoprotein which hydroxylates 2-aminobenzoyl-CoA and reduces the enzyme-bound intermediate to a nonaromatic product has been studied in some detail (16). In the case of multicomponent dioxygenases, the reduced flavin donates electrons to a specific electron-accepting iron-sulfur protein.…”

Section: CMmentioning

confidence: 99%

“…Whereas the dearomatizing reaction in case of 2-aminobenzoyl-CoA has been studied (2,8,9,10,16,23,24,41), the fate of phenylacetyl-CoA is unknown (29). A similar aerobic phenylacetyl-CoA pathway seems to exist in Pseudomonas putida (28,33), Escherichia coli (13,38), and other bacteria (29,38).…”

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confidence: 99%

Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium Azoarcus evansii

et al. 2001

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The aerobic metabolism of benzoate in the proteobacterium Azoarcus evansii was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.

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“…For both reactions, monooxygenation and reduction, enzyme-bound FAD is required, albeit with different roles. For hydroxylation, a 4a-FAD-hydroperoxide is the key intermediate; for reduction, FADH 2 transfers the hydride equivalent from NADH (17).…”

Section: Vol 183 2001 Genes Of New Anthranilate Metabolism 5275mentioning

confidence: 99%

“…The reaction requires 2 NADH molecules and 1 O 2 molecule. ACMR and the reaction it catalyzes have been studied experimentally and theoretically in some detail (12,13,17,22,23,24,30). In addition, its Nterminal amino acid sequence has been determined (2).…”

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confidence: 99%

Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium Azoarcus evansii

et al. 2001

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In the ␤-proteobacterium Azoarcus evansii, the aerobic metabolism of 2-aminobenzoate (anthranilate), phenylacetate, and benzoate proceeds via three unprecedented pathways. The pathways have in common that all three substrates are initially activated to coenzyme A (CoA) thioesters and further processed in this form. The two initial steps of 2-aminobenzoate metabolism are catalyzed by a 2-aminobenzoate-CoA ligase forming 2-aminobenzoyl-CoA and by a 2-aminobenzoyl-CoA monooxygenase/reductase (ACMR) forming 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. Eight genes possibly involved in this pathway, including the genes encoding 2-aminobenzoate-CoA ligase and ACMR, were detected, cloned, and sequenced. The sequence of the ACMR gene showed that this enzyme is an 87-kDa fusion protein of two flavoproteins, a monooxygenase (similar to salicylate monooxygenase) and a reductase (similar to old yellow enzyme). Besides the genes for the initial two enzymes, genes for three enzymes of a ␤-oxidation pathway were found. A substrate binding protein of an ABC transport system, a MarR-like regulator, and a putative translation inhibitor protein were also encoded by the gene cluster. The data suggest that, after monooxygenation/reduction of 2-aminobenzoyl-CoA, the nonaromatic CoA thioester intermediate is metabolized further by ␤-oxidation. This implies that all subsequent intermediates are CoA thioesters and that the alicyclic carbon ring is not cleaved oxygenolytically. Surprisingly, the cluster of eight genes, which form an operon, is duplicated. The two copies differ only marginally within the coding regions but differ substantially in the respective intergenic regions. Both copies of the genes are coordinately expressed in cells grown aerobically on 2-aminobenzoate.2-Aminobenzoic acid (anthranilic acid) plays an important role in the synthesis and degradation of many N-heterocyclic compounds such as tryptophan, indole, indoleacetic acid, and derived compounds. As a consequence of its wide occurrence, 2-aminobenzoate is a common substrate for many microorganisms that are able to cleave aromatic rings.There are three established aerobic pathways by which 2-aminobenzoate is converted to either catechol or gentisate (reviewed in reference 26). These compounds are central intermediates of aerobic aromatic metabolism which are further oxidized and cleaved, via either ortho-or meta-cleavage pathways. Notably, the aromatic ring is cleaved oxygenolytically, and coenzyme A (CoA) thioesters of organic acids come into play only after ring cleavage, e.g., as 3-oxo-adipyl-CoA and succinyl-CoA. Such rules also apply to the aerobic metabolism of other aromatic substrates. These pathways have been discussed and reviewed elsewhere (14).

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NIH shift in flavin-dependent monooxygenation: Mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase

Cited by 28 publications

References 22 publications

Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene

Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene

Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium Azoarcus evansii

Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium Azoarcus evansii

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