Department of Biology, Memorial University of Newfoundland, St. John's, NL, Canada.
PLoS One. 2019 Apr 23;14(4):e0215960. doi: 10.1371/journal.pone.0215960. eCollection 2019.
In Streptomyces clavuligerus, the gene cluster involved in the biosynthesis of the clinically used β-lactamase inhibitor clavulanic acid contains a gene (orf12 or cpe) encoding a protein with a C-terminal class A β-lactamase-like domain. The cpe gene is essential for clavulanic acid production, and the recent crystal structure of its product (Cpe) was shown to also contain an N-terminal isomerase/cyclase-like domain, but the function of the protein remains unknown. In the current study, we show that Cpe is a cytoplasmic protein and that both its N- and C-terminal domains are required for in vivo clavulanic acid production in S. clavuligerus. Our results along with those from previous studies allude towards a biosynthetic role for Cpe during the later stages of clavulanic acid production in S. clavuligerus. Amino acids from Cpe essential for biosynthesis were also identified, including one (Lys89) from the recently described N-terminal isomerase-like domain of unknown function. Homologues of Cpe from other clavulanic acid-producing Streptomyces spp. were shown to be functionally equivalent to the S. clavuligerus protein, whereas those from non-producers containing clavulanic acid-like gene clusters were not. The suggested in vivo involvement of an isomerase-like domain recruited by an ancestral β-lactamase related protein, supports a previous hypothesis that Cpe could be involved in a step requiring the opening and modification of the clavulanic acid core during its biosynthesis from 5S precursors.
在棒状链霉菌中，参与临床应用的β-内酰胺酶抑制剂克拉维酸生物合成的基因簇包含一个编码具有 C 端 A 类β-内酰胺酶样结构域的蛋白质的基因（orf12 或 cpe）。cpe 基因对于克拉维酸的产生是必不可少的，其产物（Cpe）的最近晶体结构显示还包含一个 N 端异构酶/环化酶样结构域，但该蛋白的功能仍不清楚。在本研究中，我们表明 Cpe 是一种细胞质蛋白，其 N 端和 C 端结构域均是棒状链霉菌中体内克拉维酸产生所必需的。我们的结果以及之前的研究结果都暗示 Cpe 在棒状链霉菌克拉维酸产生的后期阶段具有生物合成作用。还确定了 Cpe 中对生物合成至关重要的氨基酸，包括最近描述的具有未知功能的 N 端异构酶样结构域中的一个（Lys89）。其他产生克拉维酸的链霉菌属的 Cpe 同源物与棒状链霉菌的蛋白具有相同的功能，而那些来自含有克拉维酸样基因簇但不产生克拉维酸的非生产者的同源物则没有。推测这种异构酶样结构域的体内参与是由一个与祖先β-内酰胺酶相关的蛋白募集的，这支持了之前的假设，即 Cpe 可能参与克拉维酸生物合成过程中需要打开和修饰克拉维酸核心的步骤，该过程是从 5S 前体开始的。
