We have determined the three-dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase-2) at 0.16-nm resolution. SFase-2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur: J. Biochem. 220,[55][56][57][58][59][60][61]. In the present study, data were collected to approximately 0.16-nm resolution on a Rigaku R-AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined struc- The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a p barrel with six-stranded / 3 sheets and two a helices. The overall tertiary structure of SFase-2 is similar to the structures of other chyrnotrypsin-like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar threedimensional structures, indicating that a gene duplication event has occurred to produce these two domains.We predicted the tertiary structure of an acidic-amino-acid-specific proteinase on the basis of the crystal structure of SFase-2, and compared the active-site conformations of these two enzymes. We found a characteristic histidine cluster of three histidine residues in the active site of the acidicamino-acid-specific proteinase. The substrate recognition mechanism of SFase-2 may be mediated through the hydrophobic amino acid residues. However, in the acidic-amino-acid-specific proteinase, the positive charge of this histidine cluster would attract the negative charges of glutamic acid and aspartic acid.Serine proteinases have been classified into three groups on the basis of their substrate specificities, those which possess broad substrate specificity (subtilisin), those which cleave C-terminal bonds of the basic amino acids lysine and arginine (trypsin), and those which recognize negatively charged residues of glutamic acid and aspartic acid (V8 proCorrespondence to K. Kitadokoro, Shionogi Research Laboratories, Shionogi and Co., Ltd, 5-12-4 Sagisu, Fukushima-ku, Osaka, Japan 553Abbreviations. ATCC, American type culture collection; SFase-1 or SF-1, Streptomyces fradiae acidic-amino-acid-specific proteinase; SFase-2 or SF-2, proteinase with broad substrate specificity from S. fradiae.Enzyme. SFase-2, proteinase with broad substrate specificity from Streptomyces fradiae .Note. The novel structural data have been submitted to the Protein Data Base data bank under the accession number 2SFA. The numbering system in this study is based on the sequence for SFase-2 (Fig. 3).teinase from Staphylococcus aureus [2] and a glutamic-acidspecific protease from Bacillus lichenifomis [31).The three-dimensional structure of trypsin bound to various inhibitors su...