RESUMO
The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.
Assuntos
Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Agrobacterium/enzimologia , Proteínas de Bactérias/metabolismo , Betaína/análogos & derivados , Carnitina/metabolismo , Coenzima A Ligases/metabolismo , Microbiologia do Solo , Acil Coenzima A/química , Difosfato de Adenosina/química , Agrobacterium/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Betaína/química , Betaína/metabolismo , Carnitina/química , Clonagem Molecular , Coenzima A Ligases/genética , Coenzima A Ligases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
From investigation of 60 filamentous fungi, we identified Fusarium merismoides var. acetilereum, which uses 4-N-trimethylamino-1-butanol (TMA-butanol) as the sole source of carbon and nitrogen. The fungus produced NAD(+)-dependent TMA-butanol dehydrogenase (DH) when it was cultivated in medium containing TMA-butanol. The enzyme showed molecular mass of 40 kDa by SDS-PAGE and 160 kDa by gel filtration, suggesting that it is a homotetramer. TMA-butanol DH is stable at pH 7.5-9.0. It exhibits moderate stability with respect to temperature (up to 30 °C). Additionally, it has optimum activity at 45 °C and at pH 9.5. The enzyme has broad specificity to various alkyl alcohols and amino alkyl alcohols, and the carbon chains of which are longer than butanol. Moreover, the activity is strongly inhibited by oxidizing agents, carbonyl and thiol modulators, and chelating agents. This report is the first study examining TMA-butanol DH from eukaryotic microbes.