Cloning, sequencing, expression, and characterization of protealysin, a novel neutral proteinase from Serratia proteamaculans representing a new group of thermolysin-like proteases with short N-terminal region of precursor.
The gene of Serratia proteamaculans proteinase, protealysin, was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a precursor of 341 amino acids (AAs) with a significant homology to thermolysin-like proteinases (TLPs). The molecular weight of the purified mature active recombinant protein was 32 kDa, the N-terminal amino acid sequence was AKTSTGGEVI. The optimum pH for azocasein hydrolysis by protealysin was seven and it was completely inhibited by o-phenanthroline. The enzyme hydrolyzed 3-(2-furyl)acryloyl-glycyl-L-leucine amide, the standard substrate for TLPs, with k(cat)/K(m) ratio of (2.52 +/- 0.02) x 10(2) M(-1) s(-1). Protealysin maturation removes 50 AA from the N-terminus of the precursor. The removed region had no similarity with the preprosequence of thermolysin (232 AA) but was homologous to some other TLPs. These proteins shared a conserved 7-AA motif near the initial methionine. Such motif was also found in some nonproteolytic putative proteins; two of them were eukaryotic.
Demidyuk
Ilya V
IV
Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia. duk@img.ras.ru
Kalashnikov
Alexander E
AE
Gromova
Tatiana Yu
TY
Gasanov
Eugene V
EV
Safina
Dina R
DR
Zabolotskaya
Maria V
MV
Rudenskaya
Galina N
GN
Kostrov
Sergey V
SV
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D012705:Serratia / Q000201:enzymology* / Q000235:genetics; D013820:Thermolysin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D013820:Thermolysin