Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme.
We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678, (previously named Mycoplana bullata). A genomic library of M. ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M. ramosa. Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da. This is the first report of the structure of acetylpolyamine amidohydrolase. The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294. The recombinant enzyme was purified, and the enzymatic properties were characterized. Substrate specificities, Km values, and Vmax values were identical to those of the native enzyme purified from M. ramosa. In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme. This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.
Sakurada
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Japan.
Ohta
T
T
Fujishiro
K
K
Hasegawa
M
M
Aisaka
K
K
D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis / Q000235:genetics*; D001051:Apoenzymes / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism*; D000581:Amidohydrolases
D000619:Aminohydrolases; D001051:Apoenzymes; D002413:Cations, Divalent; D003035:Cobalt; D008667:Metalloproteins; D015032:Zinc; C058087:acetylpolyamine amidohydrolase; D000581:Amidohydrolases