Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581.
The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101. The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues. A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG). A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site. The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues. Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B. cereus. It also shares 73% homology with the thermostable neutral proteases of B. thermoproteolyticus and B. stearothermophilus. The zinc-binding sites and the catalytic residues are completely conserved in all four proteases. NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA. These results indicate that the enzyme is a neutral (metallo-) protease.
Kühn
S
S
Universität Hamburg, Abteilung Mikrobiologie, Germany.
Fortnagel
P
P
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D010450:Endopeptidases; D008666:Metalloendopeptidases; C065625:neutral protease, Bacillus stearothermophilus; D002118:Calcium