Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation

Abstract

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 Staphylococcus aureus, 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. SIGNIFICANCE STATEMENTThe Gram-positive pathogen Staphylococcus aureus is a leading cause of antibiotic resistance-associated death in the United States. Many antibiotics used to treat S. aureus, including the beta-lactams, target biogenesis of the essential peptidoglycan (PG) cell wall. Some hydrolases play important roles in cell wall construction and are potential antibiotic targets. The amidase LytH, which requires a protein partner, ActH, for activity, is one such hydrolase. Here, we uncover how the extracellular domain of ActH binds to LytH to stabilize metals in the active site for catalysis. This work advances our understanding of how hydrolase activity is controlled to contribute productively to cell wall synthesis.