Mills D A, Flickinger M C
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Appl Environ Microbiol. 1993 Sep;59(9):2927-37. doi: 10.1128/aem.59.9.2927-2937.1993.
The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.
通过用甲醇芽孢杆菌MGA3染色体DNA的基因组文库对营养缺陷型大肠杆菌lysA22突变体进行互补，克隆了甲醇芽孢杆菌MGA3的lysA基因。亚克隆将甲醇芽孢杆菌MGA3的lysA基因定位到一个2.3 kb的SmaI - SstI片段中。对该2.3 kb片段的序列分析表明，其开放阅读框编码一种48223 Da的蛋白质，该蛋白质与枯草芽孢杆菌（62%）和谷氨酸棒杆菌（40%）的中 - 二氨基庚二酸（DAP）脱羧酶氨基酸序列相似。氨基酸序列分析表明，细菌DAP脱羧酶、真核鸟氨酸脱羧酶和精氨酸脱羧酶之间存在几个保守区域，这表明在底物和辅因子磷酸吡哆醛5'-磷酸的定位方面存在共同的结构排列。甲醇芽孢杆菌MGA3的DAP脱羧酶被证明是一种二聚体（M(r) 86,000），亚基分子量约为50,000 Da。这种脱羧酶对赖氨酸有抑制作用（Ki = 0.93 mM），对DAP的Km为0.8 mM。抑制模式表明，这种酶在甲醇芽孢杆菌MGA3的赖氨酸高产菌株中的活性可能会限制赖氨酸的合成。
