Xu Qingping, Abdubek Polat, Astakhova Tamara, Axelrod Herbert L, Bakolitsa Constantina, Cai Xiaohui, Carlton Dennis, Chen Connie, Chiu Hsiu Ju, Chiu Michelle, Clayton Thomas, Das Debanu, Deller Marc C, Duan Lian, Ellrott Kyle, Farr Carol L, Feuerhelm Julie, Grant Joanna C, Grzechnik Anna, Han Gye Won, Jaroszewski Lukasz, Jin Kevin K, Klock Heath E, Knuth Mark W, Kozbial Piotr, Krishna S Sri, Kumar Abhinav, Lam Winnie W, Marciano David, Miller Mitchell D, Morse Andrew T, Nigoghossian Edward, Nopakun Amanda, Okach Linda, Puckett Christina, Reyes Ron, Tien Henry J, Trame Christine B, van den Bedem Henry, Weekes Dana, Wooten Tiffany, Yeh Andrew, Hodgson Keith O, Wooley John, Elsliger Marc André, Deacon Ashley M, Godzik Adam, Lesley Scott A, Wilson Ian A
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Oct 1;66(Pt 10):1354-64. doi: 10.1107/S1744309110021214. Epub 2010 Jul 27.
Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.
球形芽孢杆菌的二肽基肽酶VI和枯草芽孢杆菌的YkfC此前均被表征为高度特异性的γ-D-谷氨酰-L-二氨基酸内肽酶。蜡样芽孢杆菌的YkfC直系同源物（BcYkfC）在1.8 Å分辨率下的晶体结构显示，除了C端催化性NlpC/P60结构域外，它还包含两个N端细菌SH3（SH3b）结构域，NlpC/P60结构域在非常大的细胞壁相关半胱氨酸肽酶家族中普遍存在。结合的反应产物（L-丙氨酸-γ-D-谷氨酸）使得能够鉴定识别L-丙氨酸和γ-D-谷氨酸的保守序列和结构特征，因此为理解二肽基肽酶VI、YkfC和其他NlpC/P60结构域中观察到的底物特异性提供了一个清晰的框架。第一个SH3b结构域通过促进活性位点的形成在定义底物特异性方面发挥重要作用，使得只有具有游离N端丙氨酸的胞壁肽才被允许。YkfC亚家族的SH3b结构域中的一个保守酪氨酸与NlpC/P60结构域中一个保守酸性残基的存在相关，并且这两个残基都与丙氨酸的游离胺基相互作用。这种结构特征允许定义具有相同N端底物要求的NlpC/P60酶亚家族，包括一种先前表征的蓝藻L-丙氨酸-γ-D-谷氨酸内肽酶，其包含组装YkfC样活性位点的两个关键组分（一个与SH3b结构域相连的NlpC/P60结构域）。
