The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.
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Research Article|
January 05 2016
Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis
Ghulam Hasnain;
Ghulam Hasnain
*Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, U.S.A.
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Sanja Roje;
Sanja Roje
†Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, U.S.A.
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Na Sa;
Na Sa
†Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, U.S.A.
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Rémi Zallot;
Rémi Zallot
‡Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, U.S.A.
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Michael J. Ziemak;
Michael J. Ziemak
*Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, U.S.A.
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Valérie de Crécy-Lagard;
Valérie de Crécy-Lagard
‡Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, U.S.A.
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Jesse F. Gregory, III;
Jesse F. Gregory, III
§Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611, U.S.A.
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Andrew D. Hanson
Andrew D. Hanson
1
*Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, U.S.A.
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
July 17 2015
Revision Received:
October 30 2015
Accepted:
November 03 2015
Accepted Manuscript online:
November 04 2015
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 Authors; published by Portland Press Limited
2016
Biochem J (2016) 473 (2): 157–166.
Article history
Received:
July 17 2015
Revision Received:
October 30 2015
Accepted:
November 03 2015
Accepted Manuscript online:
November 04 2015
Citation
Ghulam Hasnain, Sanja Roje, Na Sa, Rémi Zallot, Michael J. Ziemak, Valérie de Crécy-Lagard, Jesse F. Gregory, Andrew D. Hanson; Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis. Biochem J 15 January 2016; 473 (2): 157–166. doi: https://doi.org/10.1042/BJ20150805
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