Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2.
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Research Article|
October 02 2015
A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions
Kelly A. Orlando;
Kelly A. Orlando
*Department of Biology, Immaculata University, Immaculata, PA 19345, U.S.A.
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Christine L. Iosue;
Christine L. Iosue
†Department of Biology, Villanova University, Villanova, PA 19085, U.S.A.
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Sarah G. Leone;
Sarah G. Leone
†Department of Biology, Villanova University, Villanova, PA 19085, U.S.A.
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Danielle L. Davies;
Danielle L. Davies
†Department of Biology, Villanova University, Villanova, PA 19085, U.S.A.
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Dennis D. Wykoff
Dennis D. Wykoff
1
†Department of Biology, Villanova University, Villanova, PA 19085, U.S.A.
1To whom correspondence should be addressed (email dennis.wykoff@villanova.edu).
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Publisher: Portland Press Ltd
Received:
May 27 2015
Revision Received:
August 10 2015
Accepted:
August 12 2015
Accepted Manuscript online:
August 12 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2015 Authors; published by Portland Press Limited
2015
Biochem J (2015) 471 (2): 187–198.
Article history
Received:
May 27 2015
Revision Received:
August 10 2015
Accepted:
August 12 2015
Accepted Manuscript online:
August 12 2015
Citation
Kelly A. Orlando, Christine L. Iosue, Sarah G. Leone, Danielle L. Davies, Dennis D. Wykoff; A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions. Biochem J 15 October 2015; 471 (2): 187–198. doi: https://doi.org/10.1042/BJ20150611
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