Substrate specificity of radical S-adenosyl-L-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics
Substrate specificity of radical S-adenosyl-L-methionine dehydratase AprD4 and its partner reductase AprD3 in the C3′-deoxygenation of aminoglycoside antibiotics
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DOI：
10.1038/ja.2016.110
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发表时间：
2017-04-01
影响因子：
3.3
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Kudo, Fumitaka;Tokumitsu, Takahiro;Eguchi, Tadashi
自由基S-腺苷-L-甲硫氨酸还原酶AprD 4和NADPH依赖性还原酶AprD 3负责安普霉素生物合成中假二糖巴龙胺的C3 '-脱氧。这些酶参与氨基糖苷类耐药菌株中不被3 '-磷酸转移酶修饰的特征性结构基序的构建。AprD 4通过自由基介导的反应机制催化巴龙胺的C3 '-脱水，得到4'-氧代橄榄胺，然后用NADPH将其还原，得到橄榄胺。在本研究中，这种独特的酶组合的底物特异性进行了研究。AprD 4能识别巴龙胺、新霉胺、卡那霉素C和卡那霉素B，在氨基糖苷类化合物的C3 '-脱水过程中生成5'-脱氧腺苷，但不能识别2 '-N-乙酰巴龙胺和巴龙霉素。只有巴龙胺和卡那霉素C被AprD 4和AprD 3转化为相应的C3 '-脱氧化合物。AprD 3识别4 '-氧代橄榄胺部分，包括假三糖卡那霉素C，并且似乎排斥新霉胺和卡那霉素B的C6'处的氨基。手性氘标记的NADPH被用于鉴定AprD 3在还原4 '-氧代橄榄丹明时转移NADPH的pro-S氢原子以得到橄榄丹明。
A radical S-adenosyl-L-methionine dehydratase AprD4 and an NADPH-dependent reductase AprD3 are responsible for the C3'-deoxygenation of pseudodisaccharide paromamine in the biosynthesis of apramycin. These enzymes are involved in the construction of the characteristic structural motif that is not modified by 3'-phosphotransferase in aminoglycoside-resistant bacterial strains. AprD4 catalyzes the C3'-dehydration of paromamine via a radical-mediated reaction mechanism to give 4'-oxolividamine, which is then reduced by AprD3 with NADPH to afford lividamine. In the present study, the substrate specificity of this unique combination of enzymes has been investigated. AprD4 was found to recognize paromamine, neamine, kanamycin C, and kanamycin B to afford 5'-deoxyadenosine as one of products during the C3'-dehydration of aminoglycosides, but not 2'-N-acetylparomamine and paromomycin. Only paromamine and kanamycin C were converted to the corresponding C3'-deoxygenated compounds by AprD4 and AprD3. AprD3 recognizes the 4'-oxolividamine moiety, including the pseudotrisaccharide kanamycin C, and seems to reject the amino group at C6' of neamine and kanamycin B. Chirally deuterium-labeled NADPH was used to identify that that AprD3 transfers the pro-S hydrogen atom of NADPH when reducing 4'-oxolividamine to give lividamine.