Lucher L A, Chen Y M, Walker J B
Department of Biochemistry, Rice University, Houston, Texas 77251.
Antimicrob Agents Chemother. 1989 Apr;33(4):452-9. doi: 10.1128/AAC.33.4.452.
Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which represent transaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminations were catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-fold from late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. The cofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstrated its participation in the transamination reactions of this enzyme. The major physiological amino donor for the enzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as 2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. We found that crude extracts contained omega-amidase activity, which may render transaminations with glutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted to deoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrasts with the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of the enzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participates predominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzes reactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurification of two aminotransferases cannot be ruled out, our data are consistent with the participation of a single aminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M. purpurea. We propose that this aminotransferase participates in a key initial step in the biosynthesis of most aminocyclitol antibiotics.
庆大霉素产生菌紫色小单孢菌的透析提取物催化的反应代表了2-脱氧链霉胺生物合成中所提出的转氨作用。为了确定这些转氨作用是由单一的转氨酶还是多种酶催化的，我们从紫色小单孢菌中纯化并鉴定了一种L-谷氨酰胺：酮基-scyllo-肌醇转氨酶。该酶从野生型紫色小单孢菌和一种无2-脱氧链霉胺的自养突变体的对数生长后期菌丝体中纯化了130至150倍。发现辅因子磷酸吡哆醛与该酶紧密结合，光谱分析表明其参与了该酶的转氨反应。该酶的主要生理氨基供体似乎是L-谷氨酰胺；源自谷氨酰胺的酮酸产物被鉴定为2-酮戊二酸，表明谷氨酰胺的α氨基参与了转氨作用。我们发现粗提取物含有ω-酰胺酶活性，这可能使体内与谷氨酰胺的转氨作用不可逆。转氨酶的底物特异性仅限于脱氧环醇、单氨基环醇和二氨基环醇、谷氨酰胺和2-酮戊二酸，这与哺乳动物谷氨酰胺转氨酶更广泛的底物特异性形成对比。该酶在对数生长后期出现，再加上其狭窄的底物特异性，表明它主要参与2-脱氧链霉胺的生物合成而不是一般代谢。该酶催化的反应代表了2-脱氧链霉胺生物合成的两个转氨步骤。虽然不能排除两种转氨酶共纯化的可能性，但我们的数据与单一转氨酶参与紫色小单孢菌生物合成过程中2-脱氧链霉胺两个氨基形成的情况一致。我们提出这种转氨酶参与了大多数氨基环醇抗生素生物合成的关键起始步骤。
