Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115.
Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 2022 Jun 28;119(26):e2201141119. doi: 10.1073/pnas.2201141119. Epub 2022 Jun 22.
Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in , acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.
细菌肽聚糖（PG）细胞壁的构建和重塑必须与细胞生长和分裂精细协调。细胞壁构建的核心是能够切割肽聚糖键的水解酶。这些酶也是潜在的新型抗生素靶标。肠球菌中的酰胺酶 LytH 就是这样一种水解酶，它从 PG 上除去茎肽，控制新 PG 链插入的底物位置，从而调节细胞大小。当它不存在时，细胞会过度生长并出现分裂缺陷。LytH 的活性需要一个蛋白质伴侣 ActH，它由一个细胞内结构域、一个大型菱形蛋白酶结构域和三个细胞外四肽重复（TPR）组成。在这里，我们证明 ActH 的酰胺酶激活功能完全包含在其细胞外 TPR 中。我们表明，ActH 结合稳定了 LytH 活性位点中的金属，而 LytH 金属结合反过来又需要与 ActH 稳定结合。我们进一步展示了 LytH 和 ActH 的细胞外结构域的复合物结构。我们的研究结果表明，金属辅因子的稳定化是酰胺酶激活剂普遍采用的策略，并且 ActH 在单个蛋白中具有多种功能。
