Hagemeier C H, Chistoserdova L, Lidstrom M E, Thauer R K, Vorholt J A
Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany.
Eur J Biochem. 2000 Jun;267(12):3762-9. doi: 10.1046/j.1432-1327.2000.01413.x.
Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32-kDa NADP-specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic alpha-proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 microM with NAD+ (Vmax = 1100 U x mg(-1) and 100 microM with NADP+ (Vmax = 950 U x mg(-1). The Km value for NAD+ was 200 microM and for NADP+ 20 microM. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N-terminal amino-acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.
最近发现，嗜甲基甲基杆菌AM1的细胞提取物可催化亚甲基四氢甲蝶呤（亚甲基H4MPT）与NAD +和NADP +的脱氢反应。已经报道了32 kDa的NADP特异性亚甲基H4MPT脱氢酶（MtdA）的纯化。在这里，我们报道了这种需氧α-变形杆菌中第二种亚甲基H4MPT脱氢酶（MtdB）的特性。纯化后的MtdB表观分子量为32 kDa，通过三元复合物催化机制，可催化亚甲基H4MPT与NAD +和NADP +氧化为亚胺基H4MPT。亚甲基H4MPT对NAD +的Km为50 microM（Vmax = 1100 U x mg（-1）），对NADP +的Km为100 microM（Vmax = 950 U x mg（-1））。NAD +的Km值为200 microM，NADP +的Km值为20 microM。与MtdA相反，MtdB不能催化亚甲基四氢叶酸的脱氢反应。通过N端氨基酸序列，鉴定出MtdB编码基因是位于基因簇中的orfX，其翻译产物与以前仅在产甲烷和硫酸盐还原古细菌中发现的酶具有高度的序列同一性。尽管其位置如此，MtdB与古细菌酶没有序列相似性。与MtdA的相似性最高，其编码基因位于古细菌岛之外。MtdB缺陷型突变体无法在甲醇上生长，并且对甲醛表现出明显的敏感性。基于突变体表型和动力学特性，讨论了MtdB和MtdA的可能功能。我们还报告说，MtdB和MtdA都可以在大肠杆菌中异源过量表达，从而使这两种酶易于用于结构分析。
