Ajjawi Imad, Tsegaye Yoseph, Shintani David
Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557, USA.
Arch Biochem Biophys. 2007 Mar 1;459(1):107-14. doi: 10.1016/j.abb.2006.11.011. Epub 2006 Nov 18.
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.
2-甲基-4-氨基-5-羟甲基嘧啶磷酸激酶/硫胺素单磷酸焦磷酸化酶（HMPPK/TMPPase）是硫胺素生物合成中的关键酶。在拟南芥基因组中鉴定出的一个候选HMPPK/TMPPase基因弥补了th1突变体的硫胺素营养缺陷，从而证明th1位点对应于HMPPK/TMPPase的结构基因。野生型HMPPK/TMPPase基因与th1-201突变等位基因之间的序列比较确定了一个单点突变，该突变导致在HMPPK结构域中一个保守的丝氨酸残基被苯丙氨酸取代。对大肠杆菌中突变型HMPPK/TMPPase的功能分析表明，突变酶的HMPPK结构域中的氨基酸取代导致构象变化，严重损害了双功能酶的两种活性。还进行了研究以确定叶绿体是拟南芥HMPPK/TMPPase的特定亚细胞定位。
