REACTIONS CATALYZED BY PURIFIED L-GLUTAMINE - KETO-SCYLLO-INOSITOL AMINOTRANSFERASE, AN ENZYME REQUIRED FOR BIOSYNTHESIS OF AMINOCYCLITOL ANTIBIOTICS
REACTIONS CATALYZED BY PURIFIED L-GLUTAMINE - KETO-SCYLLO-INOSITOL AMINOTRANSFERASE, AN ENZYME REQUIRED FOR BIOSYNTHESIS OF AMINOCYCLITOL ANTIBIOTICS
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DOI：
10.1128/aac.33.4.452
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发表时间：
1989-04-01
影响因子：
4.9
通讯作者：
WALKER, JB
中科院分区：
文献类型：
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作者：
LUCHER, LA;CHEN, YM;WALKER, JB
庆大霉素生产者紫色小单孢菌的透析提取物催化的反应代表了建议用于2-脱氧链胺生物合成的转氨基。为了确定这些转氨酶是由单一的转氨酶还是由多个酶催化的，我们从紫毛分枝杆菌中纯化并鉴定了一个L-谷氨酰胺：酮-水杨酸肌醇转氨酶。该酶从野生型紫云杉和无2-脱氧链胺的独生菌的对数末期菌丝体中纯化了130-150倍。辅因子吡哆醛磷酸被发现与该酶紧密结合，光谱分析表明它参与了该酶的转位反应。该酶的主要生理氨基供体似乎是L-谷氨酰胺；谷氨酰胺衍生的酮酸产物经鉴定为2-酮戊二酸，表明谷氨酰胺的α氨基参与了转氨基反应。我们发现粗提物中含有omega-amidase活性，这可能使谷氨酰胺的转氨酶在体内不可逆。氨基转移酶的底物专一性仅限于脱氧环醇、单氨基半胱醇、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专一性形成了鲜明对比。该酶在对数后期的出现，加上其狭窄的底物专一性，表明它主要参与2-脱氧链胺的生物合成，而不是一般的代谢。该酶催化的反应代表了2-脱氧链胺生物合成的两个转氨化步骤。虽然不能排除两种氨基转移酶相互作用的可能性，但我们的数据与单一的氨基转移酶参与了2-脱氧链胺的两个氨基的形成是一致的。我们认为这种氨基转移酶参与了大多数氨基环素类抗生素生物合成的关键初始步骤。
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 transmination 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.