Anaerobic Metabolism of Indoleacetate

Abstract

The anaerobic metabolism of indoleacetate (indole-3-acetic acid [IAA]) in the denitrifying betaproteobacterium Azoarcus evansii was studied. The strain oxidized IAA completely and grew with a generation time of 10 h. Enzyme activities that transformed IAA were present in the soluble cell fraction of IAA-grown cells but were 10-fold downregulated in cells grown on 2-aminobenzoate or benzoate. The transformation of IAA did not require molecular oxygen but required electron acceptors like NAD + or artificial dyes. The first products identified were the enol and keto forms of 2-oxo-IAA. Later, polar products were observed, which could not yet be identified. The first steps likely consist of the anaerobic hydroxylation of the N-heterocyclic pyrrole ring to the enol form of 2-oxo-IAA, which is catalyzed by a molybdenum cofactor-containing dehydrogenase. This step is probably followed by the hydrolytic ring opening of the keto form, which is catalyzed by a hydantoinase-like enzyme. A comparison of the proteome of IAA- and benzoate-grown cells identified IAA-induced proteins. Owing to the high similarity of A. evansii with strain EbN1, whose genome is known, we identified a cluster of 14 genes that code for IAA-induced proteins involved in the early steps of IAA metabolism. These genes include a molybdenum cofactor-dependent dehydrogenase of the xanthine oxidase/aldehyde dehydrogenase family, a hydantoinase, a coenzyme A (CoA) ligase, a CoA transferase, a coenzyme B 12 -dependent mutase, an acyl-CoA dehydrogenase, a fusion protein of an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, and a periplasmic substrate binding protein for ABC transport as well as a transcriptional regulator of the GntR family. Five predicted enzymes form or act on CoA thioesters, indicating that soon after the initial oxidation of IAA and possibly ring opening, CoA thioesters are formed, and the carbon skeleton is rearranged, followed by a CoA-dependent thiolytic release of another CoA thioester. We propose a scheme of an anaerobic IAA metabolic pathway that ultimately leads to 2-aminobenzoyl-CoA or benzoyl-CoA.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

“…The authors proposed that the degradation pathway involves the carbon skeleton rearrangement of 2-(2Ј-aminophenyl)succinyl-CoA to (2Ј-aminobenzyl)malonyl-CoA (Fig. 1f) by a novel acyl-CoA mutase and used similar bioinformatic analyses to identify the same cluster of uncharacterized mutases (18). Our understanding of substrate binding in acyl-CoA mutases now allows for a re-examination of this proposal.…”

“…Although strains carrying these mutases are not well characterized, recent studies suggest that a strain related to A. aromaticum, Azoarcus evansii, employs this operon for anaerobic degradation of indoleacetate (18). The authors proposed that the degradation pathway involves the carbon skeleton rearrangement of 2-(2Ј-aminophenyl)succinyl-CoA to (2Ј-aminobenzyl)malonyl-CoA (Fig.…”

“…Finally, it was proposed that novel acyl-CoA mutases catalyze the interconversion of 2-(2Ј-aminophenyl)-succinyl-CoA and (2Ј-aminobenzyl)malonyl-CoA and related compounds (Fig. 1f) in the anaerobic degradation of aromatic compounds such as indoleacetate (18,19) as well as the interconversion of 2-(1Ј-methylalkyl)succinyl-CoA and (2Ј-methylalkyl)malonyl-CoA (Fig. 1g) in the anaerobic degradation of alkanes (20 -23).…”

Biochemical and Genetic Bases of Indole-3-Acetic Acid (Auxin Phytohormone) Degradation by the Plant-Growth-Promoting Rhizobacterium Paraburkholderia phytofirmans PsJN

“…These primers contain a 20-bp terminal sequence homologous to the terminus of the fragment to be linked, and the sequences were combined and ligated to generate a new DNA molecule in a one-step isothermal reaction (66). Also, PCR products lacking one or two of these iac genes were amplified using primer pairs listed in Table 2: iacBIHE (primer pair 2-11), iacCD (primer pair 3-4), iacG (primer pair 5-6), and iacF (primer pair 7-8) genes were used to obtain the iac1ΔA derivative; iacA (primer pair 1-12), iacIHE (primer pair 2-13), iacCD (primer pair 3-4), iacG (primer pair 5-6), and iacF (primer pair 7-8) genes were used to produce the iac1ΔB derivative; iacABIHE (primer pair 1-15), iacG (primer pair 5-6), and iacF (primer pair 7-8) genes were used to produce the iac1ΔCD derivative; iacABIH (primer pair [1][2][3][4][5][6][7][8][9][10][11][12][13][14], iacCD (primer pair 3-4), iacG (primer pair 5-6), and iacF (primer pair 7-8) genes were used to give the iac1ΔE derivative; iacABIHE (primer pair 1-2), iacCD (primer pair 3-4), and iacG (primer pair 5-16) genes were used to create the iac1ΔF derivative; iacABIHE (primer pair 1-2), iacCD (primer pair 3-4), and iacF (primer pair 8-17) genes were used to generate the iac1ΔG derivative; iacABI (primer pair [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18], iacE (primer pair [2][3][4]…”

“…An anaerobic auxin degradation pathway has been proposed in Azoarcus evansii and Aromatoleum aromaticum (8), and complete aerobic degradation of this auxin has been reported in Bradyrhizobium (9,10); Pseudomonas (11); Burkholderia, Rhodococcus, and Sphingomonas (12); and Acinetobacter (13) species. Although two routes for aerobic IAA degradation were proposed some time ago in Bradyrhizobium japonicum (9,10), with anthranilic acid as a key intermediate, genetic and additional biochemical aspects of aerobic IAA degradation have only been recently addressed (12,14).…”