Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle
| Autor: | Tina Georgi, Nadya Kurteva-Yaneva, Michael Zahn, Roland H. Müller, Thore Rohwerder, Judith Schuster, Norbert Sträter, Ulrike Krug |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular Stereochemistry Protein Conformation Hydroxybutyrates Ether Crystallography X-Ray Substrate Specificity 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Bacterial Proteins Structural Biology Acyl binding Catalytic Domain Coenzyme A Ligases Moiety Ligase activity Molecular Biology Adenylylation Burkholderiales 030304 developmental biology chemistry.chemical_classification 0303 health sciences DNA ligase Enzyme chemistry Catalytic cycle 030217 neurology & neurosurgery |
| Zdroj: | Journal of molecular biology. 431(15) |
| ISSN: | 1089-8638 |
| Popis: | 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° 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° and 130° 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. |
| Databáze: | OpenAIRE |
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