BU Wuppertal, FB-C, Chemical Microbiology AG, Piepersberg, Wuppertal, Germany.
J Appl Microbiol. 2011 Sep;111(3):642-51. doi: 10.1111/j.1365-2672.2011.05082.x. Epub 2011 Jul 12.
The 2-deoxystreptamine-containing aminoglycoside antibiotics (AGAs) constitute the largest subgroup of the aminoglycosides. Neomycin (NEO) and lividomycin (LIV) are both representatives of the pseudo-tetrasaccharide group among the NEO-type AGAs. While NEO contains a 6'-NH(2) group, the 6'-position remains unmodified in LIV. The aim of the study was to characterize the substrate specificities of the enzymes involved in the C-6'- and C-6‴-modification in order to explain the different amination patterns.
We overproduced and purified the enzymes NeoQ (bifunctional 6'- and 6‴-oxidoreductase) and NeoB (bifunctional 6'- and-6‴-aminotransferase), which had been analysed before (Huang et al. 2007), and compared the enzymatic properties with the corresponding enzymes LivQ (postulated 6‴-oxidoreductase, 72% identity to NeoQ) and LivB (postulated 6‴-aminotransferase, 71% identity to NeoB) from the LIV pathway. By applying a newly established photometric assay, we proved that LivQ oxidized only pseudotetrasaccharidic substrates at the 6‴-position. In contrast, NeoQ accepted also the pseudodisaccharidic paromamine as a substrate and oxidized the 6'- and 6‴-positions on two different precursors of NEO. The aminotransferases LivB and NeoB both transfer NH(2) groups to the 6'-position in the precursor 6'-oxo-paromamine and to the 6‴-position of 6‴-oxo-neomycin C.
The difference in the modification pattern of NEO and LIV at their 6'-positions is based only on the difference in the substrate specificities of the oxidoreductases LivQ and NeoQ, respectively. The aminotransferases LivB and NeoB share identical biochemical properties, and both are capable to transaminate the 6' and also the 6‴-position of the tested AGAs.
Our data provide information to understand the structural variations in aminoglycosides and may be helpful to interpret variations in other natural product bisoynthesis pathways.
含 2-脱氧链霉胺的氨基糖苷抗生素（AGAs）构成氨基糖苷类抗生素的最大亚组。新霉素（NEO）和洛伐霉素（LIV）均为 NEO 型 AGAs 中的假四糖组的代表。虽然 NEO 含有 6'-NH（2）基团，但 LIV 的 6'-位未被修饰。本研究的目的是表征参与 C-6'-和 C-6‴-修饰的酶的底物特异性，以解释不同的胺化模式。
我们过量表达和纯化了以前分析过的酶 NeoQ（双功能 6'-和 6‴-氧化还原酶）和 NeoB（双功能 6'-和 6‴-氨基转移酶），并将其与相应的酶 LivQ（假定的 6‴-氧化还原酶，与 NeoQ 有 72%的同一性）和 LivB（假定的 6‴-氨基转移酶，与 NeoB 有 71%的同一性）进行了比较。通过应用新建立的比色法，我们证明 LivQ 仅在 6‴-位氧化假四糖底物。相比之下，NeoQ 还接受假二糖帕罗胺作为底物，并氧化 NEO 的两个不同前体的 6'-和 6‴-位。氨基转移酶 LivB 和 NeoB 均将 NH（2）基团转移到前体 6'-氧代帕罗胺的 6'-位和 6‴-氧代新霉素 C 的 6‴-位。
NEO 和 LIV 在其 6'-位的修饰模式差异仅基于氧化还原酶 LivQ 和 NeoQ 的底物特异性差异。氨基转移酶 LivB 和 NeoB 具有相同的生化特性，均能够转氨基化测试 AGAs 的 6'和 6‴-位。
我们的数据提供了了解氨基糖苷结构变异的信息，并可能有助于解释其他天然产物生物合成途径中的变异。
