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. 2006 Apr;188(8):2919-27.
doi: 10.1128/JB.188.8.2919-2927.2006.

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase

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Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase

Johannes Gescher et al. J Bacteriol. 2006 Apr.

Abstract

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.

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Figures

FIG. 1.
FIG. 1.
(A) The proposed new aerobic benzoate metabolic pathway in A. evansii and the conventional β-ketoadipate pathway. Compounds: 1, benzoate; 2, protocatechuate; 3, catechol; 4, β-ketoadipate; 5, β-ketoadipyl-CoA; 6, succinyl-CoA; 7, acetyl-CoA; 8, benzoyl-CoA; 9, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 10, 3,4-dehydroadipyl-CoA semialdehyde; 11, cis-3,4-dehyroadipyl-CoA; 12, trans-2,3-dehydroadipyl-CoA; 13, β-hydroxyadipyl-CoA; 14, β-hydroxyadipyl-CoA lactone. The two pathways merge at β-ketoadipyl-CoA (compound 5). (B) The aerobic benzoate oxidation gene cluster of A. evansii is experimentally shown, as well as putative functions of the gene products. The sizes of the different open reading frames are indicated beneath the arrows.
FIG. 2.
FIG. 2.
HPLC separations of [ring-14C6]-benzoyl-CoA and products resulting from its conversion with purified Box enzymes. Benzoyl-CoA (A), 2,3-dihydro-2,3-dihydroxybenzoyl-CoA (dihydrodiol) resulting from transformation of benzoyl-CoA with the oxygenase/reductase (BoxBA) (B), and 3,4-dehydroadipyl-CoA semialdehyde and formate (C) are the products resulting from the hydrolytic ring opening of the dihydrodiol as catalyzed by BoxCmal. (D) Transformation of the semialdehyde to the corresponding acid 3,4-dehydroadipyl-CoA is catalyzed by BoxDmal. The small amount of benzoate results from the thioesterase action on benzoyl-CoA.
FIG. 3.
FIG. 3.
Mass spectroscopic analysis of the new intermediate arising from the conversion of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde with BoxDmal. The spectrum shows a main peak at 892 Da that agrees well with the theoretical mass of the corresponding acid (see Discussion).
FIG. 4.
FIG. 4.
13C NMR signals of [2,3,4,5,6-13C5]3,4-dehydroadipyl-CoA (top) obtained from a reaction mixture containing [ring-13C]benzoyl-CoA, NADPH, and BoxABCD. The spectrum was recorded after protein precipitation, solid-phase extraction, and concentration of the eluent. The coupling pattern is indicated.
FIG. 5.
FIG. 5.
Partial alignment of the ALDH2 protein sequence, of the aldehyde dehydrogenases from S. mutans and V. harveyi, and of BoxD from A. evansii. Conserved amino acids which function in cofactor binding and specificity are highlighted by letters in boldface type, the catalytic cysteine residue is highlighted in black, the two conserved glutamate residues that might function in the activation of the cysteine nucleophile are highlighted in gray, and the histidine residue that is involved in the catalytic mechanism of the V. harveyi enzyme and eventually BoxD is highlighted by a capital letter.

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