A functional comparison of the TET aminopeptidases of P. furiosus and B. subtilis with a protein-engineered variant recombining the former's structure with the latter's active site

Kapoor, Divya and Singh, Balvinder and Karthikeyan, Subramanian and Guptasarma, Purnananda (2010) A functional comparison of the TET aminopeptidases of P. furiosus and B. subtilis with a protein-engineered variant recombining the former's structure with the latter's active site. Enzyme and Microbial Technology, 46 (1). pp. 1-8. ISSN 01410229

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Abstract

We have produced and characterized three microbial tetrahedral (TET) aminopeptidases: the previously uncharacterized Bacillus subtilis aminopeptidase (BsuAP), a Pyrococcus furiosus aminopeptidase (PfuAP), and a protein-engineered PfuAP-derived ‘designer’ aminopeptidase (MutAP) in which the entire active site of PfuAP is replaced with that of BsuAP through the making of 9 non-contiguous structure-guided mutations. The temperature/pH values of optimal function of MutAP (60 °C/pH 7.0) were found to be comparable to those of its progenitors, BsuAP (70 °C/pH 7.5) and PfuAP (80 °C/pH 8.0). The Km of MutAP (3.8 mM) was similar to that of PfuAP (5.0 mM) and unlike that of BsuAP (20.8 mM); however, unlike PfuAP, MutAP showed severe substrate-based inhibition like BsuAP, at substrate exceeding 5 mM concentration. MutAP thus inherits certain characteristics from each of its progenitors. At the same time, the Kcat of MutAP was ∼185-fold lower than that of PfuAP and ∼300-fold lower than that of BsuAP, indicating an unanticipated slowing down of activity. The results provide tentative evidence that ‘structure-guided transplantation’ of active sites between proteins can help in recombining enzyme characteristics in interesting and unanticipated ways, to help create novel enzymes.

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords:	Protein engineering; Enzyme engineering; Active site transplantation; Enzyme characterization
Subjects:	Q Science > QR Microbiology
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	28 Feb 2012 16:07
Last Modified:	28 Feb 2012 16:07
URI:	http://crdd.osdd.net/open/id/eprint/1120

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