Cloning and Nucleotide Sequence of the Gene Encoding a Serine Proteinase Inhibitor Named Marinostatin from a Marine Bacterium,Alteromonassp. Strain B-10-31

Abstract

The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.

Solution Structure of Marinostatin, a Natural Ester-Linked Protein Protease Inhibitor

“…As mentioned above, the Arg 5 NH of MST-2SS exchanged much faster than that of MST- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), and the Cys 8 NH of MST-2SS also exchanged faster, as compared to the corresponding residue (Ser 8 ) of MST- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The undefined mutual position between the N-and C-cyclic regions of MST-2SS should cause the fast exchange of the Cys 8 residue, because the amide proton of Ser 8 in MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) is covered with the C-cyclic region ( Figure 5). The existence of the structural difference between MST-2SS and MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)…”

“…Hydrogen-deuterium experiments of MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) showed that the backbone NH protons of Arg 5 , Tyr 6 , and Ser 8 were slowly exchanged (Figure 4): The exchange rate of Arg 5 was the slowest one, 3 × 10 -4 min -1 , and those of Tyr 6 and Ser 8 were both 2 × 10 -3 min -1 . Those exchange rates were significantly slower than those of a random coiled peptide (24).…”

“…To investigate an effect of the ester linkage on the structure, the NMR spectra of MST-2SS were analyzed by the same procedure as those of MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). A strong NOE cross-peak between Tyr 6 H R and Pro 7 H R indicative of a cis-conformation and NOE cross-peaks between Arg 5 and Cys 9 were observed for MST-2SS as well as MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, the number of NOE cross-peaks observed for MST-2SS was smaller than that for MST- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12).…”

“…The undefined mutual position between the N-and C-cyclic regions of MST-2SS should cause the fast exchange of the Cys 8 residue, because the amide proton of Ser 8 in MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) is covered with the C-cyclic region ( Figure 5). The existence of the structural difference between MST-2SS and MST (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) is also suggested by large chemical shift differences (|δ| > 0.2 ppm) of the corresponding H R protons for Met 4 , Arg 5 , Tyr 6 , and Cys 9 of MST-2SS (Table S2 in the Supporting Information), and by the 3 J HNR coupling constant difference for Met 4 and Tyr 6 . Since the H R chemical shift and 3 J HNR are indicative of the secondary structure (21), these differences suggest that these two structures are different around the reactive site in the absence of the protease.…”

“…It was reported that the disappearance of the interaction between the loop and the scaffold results in considerable enhancement of the loop mobility and reduced rigidity of the scaffold (10,27). The hydrogen bond of the ester linkage to the amide proton of the P1′ residue would be crucial for suppressing the conformational fluctuation of the fused two-cyclic structure (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) includes one hydrogen-bond restraint. The number in parentheses is that of interresidue NOEs.…”