Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508-000, Brazil.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, U.K.
ACS Chem Biol. 2024 Sep 20;19(9):2002-2011. doi: 10.1021/acschembio.4c00334. Epub 2024 Aug 29.
Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.
氨基糖苷类抗生素是治疗主要由革兰氏阴性菌引起的严重感染的重要抗生素。庆大霉素是一种氨基糖苷类抗生素，尽管具有毒性，但临床上仍用于治疗多种肺部和尿路感染。庆大霉素的商业形式是五种化合物的混合物，它们在一个氨基糖的甲基化方面存在微小差异。在两种化合物，即庆大霉素 C2 和 C2a 的情况下，唯一的区别是 C-6'上连接的甲基的立体化学。GenB2 是负责这种差向异构化的酶，是由庆大霉素生物合成基因簇编码的四个 PLP 依赖性酶之一。在此，我们已经确定了 GenB2 与 PMP 形成的全酶形式的结构，以及与底物类似物庆大霉素 X2 和 G418 形成的三元复合物的结构。基于结构分析，我们能够确定该酶催化机制的结构基础，还通过定点突变研究了该机制。史无前例的是，GenB2 是来自折叠 I 的 PLP 依赖性酶，它能够催化差向异构化，但与使用靠近 N 端的半胱氨酸残基的折叠 III PLP 依赖性差向异构酶的机制不同。用丝氨酸或丙氨酸取代该半胱氨酸残基完全消除了酶的差向异构酶功能，证实了其参与。这项研究不仅有助于理解庆大霉素生物合成的酶学，而且为探索新氨基糖苷类衍生物的酶法生产提供了有价值的细节。
