Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544

Abstract

A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDSPAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase.The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2,2,2, with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.Many studies have been done with the serine proteinases produced by Streptomycetes. Streptomyces griseus, in particular, produces many proteinases [l, 21, three of which have been well studied: proteinase A (SGPA) [3], proteinase B (SGPB) [4], and trypsin-like proteinase [5]. These enzymes have been isolated from the commercially available extracellular filtrate, termed Pronase (Kaken Seiyaku, Tokyo) ; their primary structures, biochemical properties and X-ray structures have been reported [6-81. Recently, an acidic-aminoacid-specific proteinase from S. griseus has been described [9, 101. The serine proteinases produced by S. fradiae have been described by Morihara et al. 1111 and Nickerson et al. [12].

Purification and Characterization of a Unique Alkaline Elastase from Micrococcus luteus

“…The amino acid composition of MLP was similar to the composition of alkaline proteases and subtilisins from Bacillus strains, which range in molecular mass from 23,700 to 30,000 Da (18,48) and to the composition of the 19,000-Da alkaline protease from Streptomyces fradiae (26). The greatest number of residues were found as Thr, Ser, Gln/Glu, Gly, Ala, and Val; the seven reported Bacillus enzymes, however, in contrast to MLP, contained no cysteine residues.…”

“…luteus protease was significantly inhibited by the serine enzyme inhibitor PMSF and the chymotrypsin inhibitor chymostatin. These findings indicate that MLP is a member of the chymotrypsin superfamily of the serine proteases, such as those produced by certain Streptomyces and Bacillus species (18,20,26).…”

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

“…Brayer et al reported that the S2 pocket in S. griseus proteinase A is probably a necessary prerequisite for proper substrate orientation as a result of reduced specific binding in the S1 site [29). A kinetic study of SFase-2 showed that substitution of the alanine residue by proline in the P2 position increased the k,J K,, value tenfold, to give a value similar to that of proteinases A and B from S. griseus [19]. It would be interesting to Residue numbers are given for SFase-2 (Fig.…”

Purification and Some Properties of a Keratinolytic Enzyme from an AlkaliphilicNocardiopsissp. TOA-1

“…The molecular mass of NAPase, estimated as 20 kDa (Fig. 2), was close to other proteases of actinomycetes origin such as Protease A (18 kDa) and B (19 kDa) from Streptomyces griseus, 11) SFase-2 (19 kDa) from Streptomyces fradiae, 12) and protease I(21 kDa) from Nocardiopsis dassonvillei.…”