Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis.

Abstract

A comparison of the partial amino-acid sequence of neutral protease A from Bacillus subtilis with the structure of thermolysin (EC 3.4.24.4) from Bacillus thermoproteolyticus reveals that these two proteins are homologous. Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin. This similarity provides support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability. The metalloendopeptidases comprise a group of proteolytic enzymes that share a number of functional properties; these include a pH optimum near 7.0, inhibition by chelating agents-and resistance to inactivation by diisopropylphosphofluoridate or thiol reagents (1). Such "neutral proteases" have been isolated from a wide variety of sources that include bacteria, fungi, and more recently the brush border cells of rabbit kidney (1, 2). Apart from the notable exception of the amino-acid sequence (3) and three-dimensional structure (4) of thermolysin (EC 3.4.24.4), relatively little is known concerning the structural features of the metalloendopeptidases and their relation to function.Various studies have indicated that a neutral protease isolated from the culture medium of Bacilllus subtilis NRRLB3411 appears to be quite similar to thermolysin in amino-acid composition (5), kinetic behavior (6), and susceptibility to inhibitors (1, 7), which suggests that these two enzymes might be homologous. This investigation was undertaken to develop sequence data that would clarify the possible structural interrelationships between the neutral metalloendopeptidases, and in particular demonstrate that the neutral protease A from B. subtilis and the thermostable protease, thermolysin, are homologous proteins.

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase

“…The pattern of homology is particularly evident in the regions that include structurally and functionally important residues of thermolysin, as shown in Table 2, where a comparison is also made with B. subtilis neutral protease A (14). Twelve of the 13 residues implicated in the active site of thermolysin (3) Using elastase-deficient strains of P. aeruginosa, Shad et al (27)-reported that they had cloned the elastase gene from P. aeruginosa PAO1.…”

n-Terminal Amino Acid Sequences of Neutral Proteases fromBacillus amyloliquefaciensandBacillus subtilis: Identification of a Neutral Protease Gene Cloned inBacillus subtilis

“…Moreover, determination of theN-terminal amino acid sequence of the two neutral proteases revealed that the partial sequence reported by Levy et al 2 ) was not of …”

“…The antiserum against the B. amyloliquejaciens en- liquefaciens protease (Fig. 5) coincided with the sequence for B. subti/is NRRLB3411 neutral protease reported by Levy et al 2 ) When we compared the sequence for the B. subtilis NP58 enzyme with that for the B. amyloliquefaciens, there were 4 substitutions in the 20 amino acid residues at the N-terminal (Thr-Ala at the 3rd position, Thr-Ser at the -6th, Lys-Ala at the 13th, and Ser-Pro at the 16th) from the Nterminus ( Fig. 6; these positions are underlined.).…”

“…We tried to compare this protease with that of B. amyloliquefaciens, but failed. The only information available for this task was the report by Levy et al 2 ) on the N-terminal amino acid sequence of the neutral protease from B. subtilis NRRLB3411.…”

Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581

“…anz? 'losacc.hciritic.zrs (Yoshimoto et al, 1990;Yang, 1984) and B. ani~+lo-liquefaciens (Levy et al, 1975;Vasantha et al, 1984).…”