Latent LytM at 1.3A resolution

LytM, an autolysin from Staphylococcus aureus, is a Zn2 dependent glycyl glycine endopeptidase with a characteristic HxH motif that belongs to the lysostaphin type MEROPS M23 37 of metallopeptidases. Here, we present the 1.3 crystal structure of LytM, the first structure of a lysostaphin type peptidase. In the LytM structure, the Zn2 is tetrahedrally coordinated by the side chains of N117, H210, D214 and H293, the second histidine of the HxH motif. Although close to the active site, H291, the first histidine of the HxH motif, is not directly involved in Zn2 coordination, and there is no water molecule in the coordination sphere of the Zn2 , suggesting that the crystal structure shows a latent form of the enzyme. Although LytM has not previously been considered as a proenzyme, we show that a truncated version of LytM that lacks the N terminal part with the poorly conserved Zn2 ligand N117 has much higher specific activity than full length enzyme. This observation is consistent with the known removal of profragments in other lysostaphin type proteins and with a prior observation of an active LytM degradation fragment in S. aureus supernatant. The asparagine switch in LytM is analogous to the cysteine switch in pro matrix metalloprotease

AmpH, a Bifunctional dd-Endopeptidase and dd-Carboxypeptidase of Escherichia coli▿

In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display dd-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional dd–endopeptidase and dd-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (kcat/Km) of 1,200 M−1 s−1 and 670 M−1 s−1, respectively, and removed the terminal d-alanine from muropeptides with a C-terminal d-Ala-d-Ala dipeptide. Both dd-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10−3 nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the dd-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling