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Publication Abstract from PubMed

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154 degrees rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50 degrees and 130 degrees from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle.,Zahn M, Kurteva-Yaneva N, Schuster J, Krug U, Georgi T, Muller RH, Rohwerder T, Strater N J Mol Biol. 2019 Jul 12;431(15):2747-2761. doi: 10.1016/j.jmb.2019.05.027. Epub, 2019 May 28. PMID:31145912^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.