Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.
To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611.
Manaki
0000-0002-1263-6343
M
Mimura
Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611.
Rémi
0000-0002-7317-1578
R
Zallot
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611.
Thomas D
TD
Niehaus
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611.
Ghulam
G
Hasnain
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Satinder K
SK
Gidda
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Thuy N D
TN
Nguyen
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Erin M
EM
Anderson
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Robert T
0000-0002-6915-7407
RT
Mullen
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
Greg
G
Brown
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
Alexander F
AF
Yakunin
Microbiology and Cell Science Department, University of Florida, Gainesville, Florida 32611.
Valérie
V
de Crécy-Lagard
Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611.
Jesse F
JF
Gregory
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611 adha@ufl.edu drm@ufl.edu.
Donald R
0000-0001-8694-5117
DR
McCarty
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611 adha@ufl.edu drm@ufl.edu.
Andrew D
0000-0003-2585-9340
AD
Hanson
D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
D029681:Arabidopsis Proteins; D010744:Phosphoric Monoester Hydrolases; D013835:Thiamine Pyrophosphate
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