Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1.
2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase.
Ajjawi
Imad
I
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA.
Tsegaye
Yoseph
Y
Shintani
David
D
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002736:Chloroplasts; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013831:Thiamine / Q000378:metabolism*; D013834:Thiamine Pyrophosphatase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D029681:Arabidopsis Proteins; C108436:4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); D013834:Thiamine Pyrophosphatase; D013831:Thiamine