利用拉马钱德兰图提高细菌甲酸脱氢酶的热稳定性。

Use of Ramachandran plot for increasing thermal stability of bacterial formate dehydrogenase.

作者信息

Serov A E, Odintzeva E R, Uporov I V, Tishkov V I

机构信息

Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119992, Russia.

出版信息

Biochemistry (Mosc). 2005 Jul;70(7):804-8. doi: 10.1007/s10541-005-0187-z.

DOI:10.1007/s10541-005-0187-z

PMID:16097945

Abstract

From analysis of Ramachandran plot for NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH, EC 1.2.1.2), five amino acid residues with non-optimal values phi and psi have been located in beta- and pi-turns of the FDH polypeptide chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify their role in the enzyme stability, the residues were replaced with Gly by means of site-directed mutagenesis. The His263Gly mutation caused FDH destabilization and a 1.3-fold increase in the monomolecular inactivation rate constant. The replacements Ala191Gly and Asn234Gly had no significant effect on the stability. The mutations Asn136Gly and Tyr144Gly resulted in higher thermal stability and decreased the inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing effect of the Tyr144Gly mutation was shown to be additive when introduced into the previously obtained mutant FDH with enhanced thermal stability.

摘要

通过对甲基营养型细菌假单胞菌101（FDH，EC 1.2.1.2）中依赖NAD +的甲酸脱氢酶的拉氏构象图进行分析，在FDH多肽链的β-转角和π-转角中定位了5个具有非最佳值φ和ψ的氨基酸残基，例如Asn136、Ala191、Tyr144、Asn234和His263。为了阐明它们在酶稳定性中的作用，通过定点诱变将这些残基替换为甘氨酸。His263Gly突变导致FDH不稳定，单分子失活速率常数增加了1.3倍。Ala191Gly和Asn234Gly的替换对稳定性没有显著影响。Asn136Gly和Tyr144Gly突变导致热稳定性更高，失活速率分别降低了1.2倍和1.4倍。当将Tyr144Gly突变引入先前获得的具有增强热稳定性的突变体FDH中时，其稳定作用表现为累加效应。