Grabowska Maja, Jagielska Elzbieta, Czapinska Honorata, Bochtler Matthias, Sabala Izabela
International Institute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw, Poland.
Institute of Biochemistry and Biophysics, Pawinskiego 5a, 02-106 Warsaw, Poland.
Sci Rep. 2015 Oct 6;5:14833. doi: 10.1038/srep14833.
LytM is a Staphylococcus aureus autolysin and a homologue of the S. simulans lysostaphin. Both enzymes are members of M23 metallopeptidase family (MEROPS) comprising primarily bacterial peptidoglycan hydrolases. LytM occurs naturally in a latent form, but can be activated by cleavage of an inhibitory N-terminal proregion. Here, we present a 1.45 Å crystal structure of LytM catalytic domain with a transition state analogue, tetraglycine phosphinate, bound in the active site. In the electron density, the active site of the peptidase, the phosphinate and the "diglycine" fragment on the P1' side of the transition state analogue are very well defined. The density is much poorer or even absent for the P1 side of the ligand. The structure is consistent with the involvement of His260 and/or His291 in the activation of the water nucleophile and suggests a possible catalytic role for Tyr204, which we confirmed by mutagenesis. Possible mechanisms of catalysis and the structural basis of substrate specificity are discussed based on the structure analysis.
LytM是一种金黄色葡萄球菌自溶素，是模仿葡萄球菌溶葡萄球菌素的同源物。这两种酶都是M23金属肽酶家族（MEROPS）的成员，该家族主要由细菌肽聚糖水解酶组成。LytM以潜伏形式天然存在，但可通过切割抑制性N端前区而被激活。在此，我们展示了LytM催化结构域与过渡态类似物四甘氨酸次膦酸结合在活性位点的1.45Å晶体结构。在电子密度图中，肽酶的活性位点、次膦酸以及过渡态类似物P1'侧的“二甘氨酸”片段都非常清晰。配体P1侧的密度则差得多甚至没有。该结构与His260和/或His291参与水亲核试剂激活的情况一致，并暗示了Tyr204可能的催化作用，我们通过诱变证实了这一点。基于结构分析讨论了可能的催化机制和底物特异性的结构基础。
