Elucidation of structure and reaction mechanism of serratia marcescens metalloprotease inhibitor

Abstract

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). As an initial step to elucidate the structure-function relationship of SmaPI, three-dimensional structure of SmaPI was computer modelled using the structure of SMP - Erwinia chrysanthemi inhibitor as template. The secondary structure elements and overall folding are very similar. To express and secrete the SmaPI in Bacillus subtilis, Bacillus expression vector, pSM704, was constructed. The DNA fragment containing the coding sequence for SmaPI was amplified and the amplified-DNA product was inserted into the downstream region of subtilisin signal sequence. The recombinant SmaPI expressed in B. subtilis DB431 was secreted into the culture medium in a large amount. After cultivation for 32 h, the amount of SmaPI secreted into the culture medium reached about 100 mg/liter when estimated by measuring inhibitory activities toward SMP. The $NH_2$-terminal amino acid sequencing analysis confirmed that authentic SmaPI and the recombinant SmaPI have the same $NH_2$-terminal amino acid sequences. The inhibitory activity of the purified recombinant SmaPI was found to be nearly equivalent to that of authentic SmaPI. In sequential deletion analysis of the $NH_2$-terminal region of the SmaPI, SmaPIs starting at Ser-2 and Leu-3 residues, respectively, had nearly a full inhibitory activity toward SMP. However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity. Furthermore, kinetic analysis demonstrated that SmaPI starting at the Ala-4 residue had an inhibition constant for SMP approximately 4-fold higher than that of wild-type SmaPI. The interactions of Leu-3 with SMP contribute $0.73kcal mol^{-1}$ to the overall stability of the SMP - SmaPI complex ($8.44kcal mol^{-1}$). To elucidate the detailed role of the Leu-3 residue in inhibitory activity of SmaPI, several site-directed mutations were introduced. The inhibitory act...