Two different alanine dehydrogenases from Geobacillus kaustophilus: their biochemical characteristics and differential expression in vegetative cells and spores
Two different alanine dehydrogenases from Geobacillus kaustophilus: their biochemical characteristics and differential expression in vegetative cells and spores
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嗜热地芽孢杆菌两种不同的丙氨酸脱氢酶：它们的生化特征以及在营养细胞和孢子中的差异表达
DOI：
10.1016/j.bbapap.2023.140904
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
T. Ohshima
中科院分区：
文献类型：
--
作者：
M. Maeno;T. Ohmori;D. Nukada;H. Sakuraba;T. Satomura;T. Ohshima
在嗜热芽孢杆菌基因组中发现了两个可能的丙氨酸脱氢酶基因(GK2752和GK3448)，由这两个基因推导的氨基酸序列具有很高的同源性(71%)，基于这两个可能的丙氨酸脱氢酶的氨基酸序列和同源蛋白的系统发育树表明，这两个可能的ALADH基因(GK2752和GK3448)属于不同的类群。两种重组基因产物均表现出较高的NAD+依赖的aladh活性，并进行了纯化和详细鉴定。两种酶对低、高PHS和高温(70℃)均表现出较高的稳定性。动力学分析表明，两种酶的活性均按相同的有序BiTer机理进行。X-射线晶体分析表明，这两个AlaDH具有相似的同六面体结构。值得注意的是，GK3448-ALADH在G的营养细胞中被检测到。GK2752-aladh只存在于孢子中，而不存在于孢子中。这是首次报道在营养细胞和孢子中分别表达了两个AlaDh。
Two putative alanine dehydrogenase (AlaDH) genes (GK2752andGK3448) were found in the genome of a thermophilic spore-forming bacterium,Geobacilluskaustophilus.The amino acid sequences deduced from the two genes showed mutually high homology (71%), and the phylogenetic tree based on the amino acid sequences of the two putative AlaDHs and the homologous proteins showed that the two putative AlaDH genes (GK2752andGK3448) belong to different groups. Both of the recombinant gene products exhibited high NAD+-dependent AlaDH activity and were purified to homogeneity and characterized in detail. Both enzymes showed high stability against low and high pHs and high temperatures (70 °C). Kinetic analyses showed that the activities of both enzymes proceeded according to the same sequentially ordered Bi-Ter mechanism. X-ray crystallographic analysis showed the two AlaDHs to have similar homohexameric structures. Notably, GK3448-AlaDH was detected in vegetative cells ofG. kaustophilusbut not spores, while GK2752-AlaDH was present only in the spores. This is the first report showing the presence of two AlaDHs separately expressed in vegetative cells and spores.