Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction

Abstract

The tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B₁₂-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO₂ by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.

Keywords: 2-hydroxyacyl-CoA lyase; Mycolicibacterium; acyloin condensation; degradation pathway; fuel oxygenate; isobutene; tert-butyl alcohol.

FIGURE 1

Bacterial conversion of tert-butyl compounds. (A) Proposed pathways for the degradation of fuel oxygenate ethers and isobutene via MPD and 2-HIBA. (B) Examples for gene clusters with mpdBC and/or hcl genes encoding the enzymes for the two-step oxidation of MPD to 2-HIBA and its activation to 2-hydroxyisobutyryl-CoA, respectively. The magenta colored genes from strain DSM 45062 and in the clusters of strains EC080625-04 and ACPA4 correspond to proteins WP_018331913.1, ALE72461.1 and WP_095888858.1, respectively, which likely function as TPP-dependent 2-hydroxyacyl-CoA lyases, for example, for the cleavage of 2-hydroxyisobutyryl-CoA to acetone and formyl-CoA. GMC family, glucose-methanol-choline oxidoreductase family.

FIGURE 2

Metabolites of tert-butyl compound conversion and selenium dependence of formic acid removal in strain A. chiangmaiensis DSM 45062. Cultures degrading (A) MPD and (B) 2-HIBA as main carbon source when incubated in mineral salt medium not supplemented with selenium. (C) Gene clusters encoding selenocysteine-containing FDHs and associated genes. Predicted position of the selenocysteine in the corresponding FdhA subunits and the protein length are indicated. (D) Molar ratio of formic acid formation versus 2-HIBA consumption in fed-batch cultures with 2-HIBA as main carbon source incubated in mineral salt medium with or without selenium supplementation.

FIGURE 3

Induction of (A) the lyase-HCL and (B) the Mim gene clusters in fed-batch cultures of A. chiangmaiensis DSM 45062 grown on 2-HIBA or acetone in mineral salt medium supplemented with selenium. For comparison, abundance of proteins encoded by genes directly up- and downstream of the clusters is shown as well.

FIGURE 4

Proposed reaction steps for the degradation of the 2-hydroxyisobutyryl-CoA cleavage products acetone and formyl-CoA in strain A. chiangmaiensis DSM 45062. Likely, hydroxylation of acetone and the subsequent acetol oxidation to methylglyoxal are catalyzed by the Mim cluster diiron monooxygenase and alcohol dehydrogenase Adh1 (WP_018330678.1), respectively. Only in 2-HIBA-grown cells, alcohol oxidation may also involve MpdB and methylglyoxal could be oxidized by MpdC. By contrast, the aldehyde oxidation is likely taken over by dehydrogenase WP_018333199.1 when cells are incubated on acetone. Formyl-CoA may be hydrolyzed to formic acid, which can be oxidized by 2-HIBA-induced cytoplasmic NAD⁺-dependent formate dehydrogenase FDH3.

FIGURE 5

2-Hydroxyisobutyryl-CoA lyase activity in strain A. chiangmaiensis DSM 45062. (A) TPP-dependent acetone formation from 2-hydroxyisobutyryl-CoA in crude extracts of cells grown on main carbon sources as indicated. The lyase activity obtained from 2-HIBA-grown cells amounts to 2.9 ± 0.1 nmol min^–1 mg protein^–1, while it is absent when the strain was incubated on acetone or glucose. (B) Mechanism for the reverse acyloin condensation of 2-hydroxyisobutyryl-CoA proposed to be catalyzed by the lyase from strain DSM 45062. The TPP-dependent reaction starts by deprotonation of the cofactor to yield the active ylide form. For simplicity, only the thiazole ring of TPP is shown.