Institute of Multidisciplinary Research for Advanced Materials, Tohoku University* Graduate School of Agricultural Science, Tohoku University**
○Hirofumi Kurokawa* Youhei Yamagata**
Subtilisin ALP I is produced from alkalophilic Bacillus NKS-21 and shows sequence similarity to both the neutral and the alkaline subtilisins. The enzyme is highly sensitive to high alkaline conditions, and the substrate specificity is different from those of well-known subtilisins, such as subtilisin BPN', Carlsberg or Savinase. To characterize its structure and function relationship, subtilisin ALP I is crystallized by batch method in two different space group, I4 with unit-cell parameters a = b = 103.6 A, c = 47.5 A and P21 with unit-cell parameters a = 45.6 A, b = 52.2 A, c = 90.1 A, β= 115.2 degree. I4 and P21 crystals diffract to 1.3 A, and 0.66 A resolution, respectively. Initial structure was solved by molecular replacement method by using the coordinates of subtilisin from Bacillus sp. KSM-K16 (PDB code 1MPT) as a search model by using the data of I4 crystal. One molecule were found in an asymmetric unit. Using the partially refined model, the structure of P21 form ALP I were also determined by molecular replacement method and two molecules were found in an asymmetric unit. Subatomic (0.66 A) resolution data of P21 crystal were measured on beamline BL5A at the Photon Factory (Tsukuba, Japan) using a Quantum 315 CCD detector. Four data scans with different exposure time were performed with single crystal. Total of 3,119,665 reflections were merged and scaled by HKL2000 and 783,190 unique reflections were obtained. The refinement were performed by SHELXL. Rcryst and Rfree are 0.101 and 0.114, respectively. Most of hydrogen atoms and possible residual valence electron density were observed in hydrogen omit map.