Crystal Structural and Functional Analysis of the Putative Dipeptidase fromPyrococcus horikoshiiOT3

Abstract

The crystal structure of a putative dipeptidase (Phdpd) fromPyrococcus horikoshiiOT3 was solved using X-ray data at 2.4 Å resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of theα/βfold, and the C-terminal domain consists of five long mixed strands, four helices, and two310helices. The structure ofPhdpd is quite similar to reported structures of prolidases fromP. furiosus(Zn-Pfprol) andP. horikoshii(Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However,Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) ofPhdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those ofPhdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein withZn2+ions is inactive.