title: Unveiling aminopeptidase P from Streptomyces lavendulae: molecular cloning, expression and biochemical characterization.
abstract: Presence of proline residues in the second position of the N-terminus in peptides restricts the usage of many aminopeptidases; however, aminopeptidase P (APP) is capable of removing this blockage. Based on the N-terminal amino acid sequences of APP from Streptomyces lavendulae, app gene was cloned in pET28a(+) and over expressed as a His-tagged protein with a molecular weight of ≈60 kDa in Escherichia coli BL21 (DE3). Nucleotide sequencing revealed a 1467 bp open reading frame encoding 488 amino acids (NCBI Accession No: GenBank: KC292272.1). The substrate specificity of the recombinant APP was analyzed by the hydrolysis of the Xaa-Pro bond in Gly-Pro dipeptide and bradykinin. K(m) and V(max) of the enzyme were found to be 0.4697 mmol l⁻¹ and 0.6396 μmol min⁻¹, respectively. APP activity was enhanced in the presence of metal ions such as Co²⁺, Mn²⁺, Mg²⁺ and Cu²⁺ ions and was inhibited by 1,10-phenanthroline, EDTA, PMSF and DTT. The atomic absorption studies revealed the presence of Mn²⁺ in the protein as a co-factor. This substrate specific metalloenzyme was found to be a tetramer and optimally active at pH 8 and 37 °C.
authors.affiliation: Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST) CSIR, Trivandrum, Thiruvananthapuram 695 019, Kerala, India.
authors.forename: Arya S
authors.initials: AS
authors.lastname: Nandan
authors.affiliation: Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST) CSIR, Trivandrum, Thiruvananthapuram 695 019, Kerala, India. Electronic address: madhavan85@hotmail.com.
authors.forename: Kesavan Madhavan
authors.initials: KM
authors.lastname: Nampoothiri
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002412:Cations; D003001:Cloning, Molecular; D004926:Escherichia coli; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase
keywords: Aminopeptidase P; Bradykinin; Gly-Pro-pNA; Metalloenzyme; Proline aminopeptidase; Streptomyces lavendulae