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 B 12-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 CO 2 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.

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

“…The AcHACL WT gene sequence was previously cloned into the pASG-IBA43 vector (IBA Lifesciences, Goettingen, Germany) containing an N-terminal His-tag and a Cterminal Strep-tag (19). AcHACL was expressed in E. coli Lemo21 (DE3) (NEB).…”

“…However, the underlying pathway of the in vivo synthesis of 2-HIBA and 2-HIB-CoA still remains elusive. In this context, a novel 2-HIBA degradation pathway was recently discovered in the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062, which contains an enzyme homolog for 2-HIBA-CoA ligase but no homolog for 2-HIB-CoA mutase ( 19 ). Instead, a HACL enzyme (AcHACL) cleaves 2-HIB-CoA into acetone and formyl-CoA.…”

“…Instead, a HACL enzyme (AcHACL) cleaves 2-HIB-CoA into acetone and formyl-CoA. Moreover, 2-butanone is formed from 2-hydroxy-2-methylbutyryl-CoA at comparable rates ( 19 ). In contrast to HsHACL1, HsHACL2, and RuHACL, which preferentially use long-chain substrates that are not relevant in biotechnology, AcHACL’s native substrate for the synthase reaction is the relatively short acetone, thereby offering great potential for the design of formate assimilation reactions.…”

Biocatalytic One-Carbon Transfer – A Review

“…In 2020, Rohwerder and co-workers postulated that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-hydroxyisobutyric acid and its precursor 2-methylpropane-1,2-diol to acetone and formyl-CoA by employing a ThDP-dependent lyase. 224 Recently, Gonzalez and co-workers pioneered the characterization of the ThDP-dependent HACL-catalyzed ligation reaction with formyl-CoA as C 1 donor substrate and unbranched, aliphatic carbonyls as acceptor substrates. 54 Formaldehyde, propanal, and nonanal, as well as the ketone acetone, were converted as acceptor substrates (Scheme 29a).…”

“…In 2020, Rohwerder and co-workers postulated that the actinobacterial­ strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-hydroxyisobutyric acid and its precursor 2-methylpropane-1,2-diol to acetone and formyl-CoA by employing a ThDP-dependent lyase. 224…”