Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia.
The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.
Neumann
S
S
Institute of Enzyme Technology, Heinrich-Heine University Düsseldorf, 52426, Jülich, Germany.
Kula
M-R
MR
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D020615:Stenotrophomonas maltophilia / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D015342:DNA Probes; D004791:Enzyme Inhibitors; D000581:Amidohydrolases