Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens

Abstract

ATCC 27117 produced very small amounts (0.8 U ml ؊1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25؇C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml ؊1 that is also localized in the periplasm of cells at 25؇C. However, a large amount of SmaPI (86 U ml ؊1) was extracellularly produced at the supraoptimal growth temperature of 37؇C from the recombinant S. marcescens(pSP2). We purified SmaPI from the culture supernatant of S. marcescens(pSP2) grown at 37؇C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.

RTX proteins: a highly diverse family secreted by a common mechanism

“…Similar inhibitors have been characterized for other RTX proteases, such as P. aeruginosa ( Feltzer et al , 2000 ), S. marcescens ( Kim et al , 1995 ) and Photorhabdus ( Valens et al , 2002 ; Bowen et al , 2003 ;). The SmaPI inhibitor of S. marcescens , however, shows a very high protease specificity, while the protease inhibitor of P. aeruginosa (APRin) exhibits a significantly higher inhibitory activity ( K D of 4 pM) compared with the inhibitors of E. chrysanthemi and S. marcescens ( K D values from 1 to 10 μM).…”

Occurrence of a metalloprotease (thermolysin) inhibitor among Brevibacillus species and purification of such inhibitor from Brevibacillus reuszeri IFO 15719T

“…The estimated molecular weight of the inhibitor produced by E. chrysanthemi against S. marcescens metalloprotease is 10 kDa 12 . The protein inhibitor (Sma PI) from S. marcescens ATCC 27117 is a monomeric, heat‐stable protein with a molecular weight of 10 kDa 11 . However, thermolysin is a thermostable neutral metallo‐endopeptidase with a molecular weight of 34.5 kDa 14 and metalloproteases from S. marcescens have a molecular weight of 50 kDa, Moreover, Sma PI inhibits SMP at a molecular ratio of 1 : 1 11 .…”

“…The protein inhibitor (Sma PI) from S. marcescens ATCC 27117 is a monomeric, heat‐stable protein with a molecular weight of 10 kDa 11 . However, thermolysin is a thermostable neutral metallo‐endopeptidase with a molecular weight of 34.5 kDa 14 and metalloproteases from S. marcescens have a molecular weight of 50 kDa, Moreover, Sma PI inhibits SMP at a molecular ratio of 1 : 1 11 . Therefore, the molecular weight of the inhibitor from strain IFO 15719 T is significantly larger than those of inhibitors from other bacteria.…”

“…Brevibacillus formosus IAM 13420 T and B. laterosporus IAM 15654 T were obtained from the IAM culture collection (The University of Tokyo, Tokyo, Japan), while B. reuszeri IFO 15719 T , B. centrosporus IFO 15540 T , B. brevis IFO 15304 T , B. borstelensis IFO 15714 T , B. choshinensis IFO 15518 T , B. parabrevis IFO 15654 T and B. agri IFO 15538 T were from IFO (Institute for Fermentation, Osaka, Japan). Thermolysin inhibitory activity in the secondary screening was assayed using azocasein 11 . Supernatant (50 µL) from each broth culture was mixed with the same volume of thermolysin solution (10 µg/mL).…”

“…In contrast, Serratia marcescens ATCC 27117 was reported to produce a protein inhibitor (Sma PI) against metalloproteases (SMP) from S. marcescens. 10 Sma PI was also characterized in detail by biochemical and molecular techniques 11 . It has also been reported that Erwinia chrysanthemi produces an inhibitor against SMP 12 …”