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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

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title 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
 
Creator Kapoor, Divya
Singh, Balvinder
Karthikeyan, Subramanian
Guptasarma, Purnananda
 
Subject QR Microbiology
 
Description 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.
 
Publisher Elsevier Science
 
Date 2010
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/1120/1/guptasarma2010.1.pdf
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
 
Relation http://dx.doi.org/10.1016/j.enzmictec.2009.09.003
http://crdd.osdd.net/open/1120/