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嗜甲基甲基杆菌AM1中依赖烟酰胺腺嘌呤二核苷酸磷酸的亚甲基四氢甲蝶呤脱氢酶

The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1.

作者信息

Vorholt J A, Chistoserdova L, Lidstrom M E, Thauer R K

机构信息

Max-Planck-Institut für terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, 35043 Marburg, Germany

出版信息

J Bacteriol. 1998 Oct;180(20):5351-6. doi: 10.1128/JB.180.20.5351-5356.1998.

Abstract

An NADP-dependent methylene tetrahydromethanopterin (H4MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO2 in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H4MPT with NADP+ rather than with NAD+, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H4F) with NADP+. With methylene H4F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (Vmax/Km) were approximately 20-fold lower than with methylene H4MPT. Whereas the dehydrogenation of methylene H4MPT (E0 = -390 mV) with NADP+ (E0 = -320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H4F (E0 = -300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H4F dehydrogenases from other bacteria and eucarya and with those of methylene H4MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).

摘要

最近有人提出,一种依赖烟酰胺腺嘌呤二核苷酸磷酸(NADP)的亚甲基四氢甲蝶呤(H4MPT)脱氢酶参与了嗜甲基甲基杆菌AM1中甲醛氧化为二氧化碳的过程。我们在此报告了这种新型酶的纯化,使其达到了表观均一性。通过N端氨基酸序列,确定它是mtdA基因的产物。纯化后的酶催化亚甲基H4MPT与NADP+而非NAD+发生脱氢反应,比活性约为400 U/mg蛋白质。它还催化亚甲基四氢叶酸(亚甲基H4F)与NADP+发生脱氢反应。然而,以亚甲基H4F为底物时,比活性(26 U/mg)和催化效率(Vmax/Km)比以亚甲基H4MPT为底物时低约20倍。亚甲基H4MPT(E0 = -390 mV)与NADP+(E0 = -320 mV)的脱氢反应基本不可逆,而亚甲基H4F(E0 = -300 mV)的脱氢反应则完全可逆。将嗜甲基甲基杆菌AM1中依赖NADP的脱氢酶的一级结构与其他细菌和真核生物中的亚甲基H4F脱氢酶以及产甲烷古菌中的亚甲基H4MPT脱氢酶的一级结构进行比较,发现只有微弱的相似性(<15%)。

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