Please use this identifier to cite or link to this item: http://localhost:8080/xmlui/handle/123456789/1820
Title: Biochemical and structural analysis of a site directed mutant of manganese dependent aminopeptidase P from Streptomyces lavendulae
Authors: Arya Nandan
Nampoothiri, K M
Keywords: Aminopeptidase P
Catalytic domain
Metalloprotease
Site directed mutagenesis
Streptomyces lavendulae
Issue Date: 2015
Publisher: Plovdiv University Press
Citation: Journal of BioScience and Biotechnology 4(2):109-116;2015
Abstract: Aminopeptidase P (APP) removes N-terminal amino acids from peptides and proteins when the penultimate residue is proline. To understand the structure-function relationships of aminopeptidase P of Streptomyces lavendulae, a conserved arginine residue was replaced with lysine (R453K) by site-directed mutagenesis. The overexpressed wild and mutant enzymes were of nearly 60 kDa and purified by nickel affinity chromatography. Kinetic analysis of R453K variant using Gly-Pro-pNA as the substrate revealed an increase in Km with a decrease in Vmax, leading to overall decrease in the catalytic efficiency, indicating that the guanidinium group of arginine plays an important role in substrate binding in APP. We constructed three dimensional models for the catalytic domains of wild and mutant enzyme and it revealed an interaction in R453 of native enzyme through hydrogen bonding with the adjacent residues making a substrate binding cavity whereas K453 did not participate in any hydrogen bonding. Hence, R453 in APP of S. lavenduale must be playing a critical role in the hydrolysis of the substrate.
URI: http://ir.niist.res.in:8080/jspui/handle/123456789/1820
ISSN: 1314-6246
Appears in Collections:2015

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