Crystal Structure Analysis of a Serine Proteinase from Streptomyces fradiae at 0.16‐nm Resolution and Molecular Modeling of an Acidic‐amino‐acid‐specific Proteinase

Abstract

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...

Glutamyl endopeptidase of Bacillus intermedius, strain 3‐19

“…According to X-ray crystallography data, their tertiary structure seems to be related to that common for chymotrypsin family [11]. It was suggested that an unusual array of three histidine residues in Streptomyces griseus protease is responsible for its specific interaction with y-carboxylate of the substrate glutamyl residue [12]; albeit, no homologous, His residues were detected in other glutamyl endopeptidases.…”

A new serine protease family with elastase activity is produced by Streptomyces bacteria

“…In mammals, there are several different types of elastases, including chymotrypsin-like elastase family member 1 in the pancreas (CELA1), neutrophil elastase and macrophage metalloprotease [5,9,10]. Elastolytic activities have also been found in some microorganisms, including Pseudomonas, Clostridium, Vibrio, Aeromonas, Bacillus, Streptomyces and Aspergillus [7,8,[11][12][13][14][15][16][17][18][19][20]. The elastases produced by Pseudomonas aeruginosa [11], Clostridium histolyticum [12], Vibrio cholera [21], Vibrio vulnificus [13], Aeromonas hydrophila [14] and Aspergillus fumigatus [7] are metalloproteases, which may play an important role in helping bacterium invasiveness and establishment of infection [7,[12][13][14].…”

“…The elastases produced by Pseudomonas aeruginosa [11], Clostridium histolyticum [12], Vibrio cholera [21], Vibrio vulnificus [13], Aeromonas hydrophila [14] and Aspergillus fumigatus [7] are metalloproteases, which may play an important role in helping bacterium invasiveness and establishment of infection [7,[12][13][14]. The elastases from Bacillus and Streptomyces species are serine proteases [15][16][17][18][19][20]. Tsai et al have reported that Bacillus subtilis produces an alkaline elastase (subtilisin YaB), which has high elastolytic activity and belongs to the subtilisin subfamily [15,22,23].…”

Isolation and characterisation ofBacillus amyloliquefaciens H2 glutamyl endopeptidase that is secreted in stationary phase of culture growth

“…These enzymes have been purified to homogeneity and extensively studied. For some enzymes, such as those from Staphylococcus epidermidis, Staphylococcus aureus, Streptomyces fradiae, Streptomyces griseus, Bacillus licheniformis, Bacillus subtilis, Bacillus intermedius the corresponding genes have been cloned and the enzymes' tertiary structures have been solved (Drapeau, 1978;Svendsen et al, 1991;Barbosa et al, 1993;Neinaber et al, 1993;Kitadokoro et al, 1994;Rebrikov et al, 1999;Ohara-Nemoto et al, 2002;Mejers et al, 2004).…”