The thiM locus and its relation to phosphorylation of hydroxyethylthiazole in Escherichia coli

Abstract

A mutant of Escherichia coli lacking hydroxyethylthiazole kinase (EC 2.7.1.50) was produced by a further mutation of a temperature-sensitive, auxotrophic mutant for hydroxyethylthiazole. The parent cells possessed two distinct enzymes capable of phosphorylating hydroxyethylthiazole: one was hydroxyethylthiazole kinase, and the other was a phosphotransferase species that required p-nitrophenylphosphate as a phosphoryl donor. Osmotic shock fluid prepared from the mutant cells phosphorylated hydroxyethylthiazole to an extent comparable to that observed with shock fluid from the parent cells, whereas extracts from shocked cells were unable to catalyze the kinase reaction. Shock fluid from a mutant of the other type obtained as a reduced phosphatase activity against p-nitrophenylphosphate did not show any appreciable activity for the phosphotransferase reaction, while extracts from shocked cells showed full kinase activity. The former mutant had lost its ability to grow on hydroxyethylthiazole at high temperature, but the latter mutant still responded to it. It thus appears that the kinase is an enzyme which might play a role in the biosynthesis of thiamine PP1 in situ. By conjugation and P1 transduction, a gene governing hydroxyethylthiazole kinase activity, for which we propose the designation thiM, was mapped on the chromosome close to thiD, a gene specifying phosphomethylpyrimidine kinase activity.The first two papers of this series (14, 15) provided evidence that five enzymes are involved in the overall reaction of thiamine PP1 biosynthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and 4-methyl-5-p-hydroxyethylthiazole (hydroxyethylthiazole) in

Characterization of Two Kinases Involved in Thiamine Pyrophosphate and Pyridoxal Phosphate Biosynthesis in Bacillus subtilis : 4-Amino-5-Hydroxymethyl-2-Methylpyrimidine Kinase and Pyridoxal Kinase

“…Thiamine from the growth medium is either phosphorylated by thiamine kinase or pyrophosphorylated by thiamine pyrophosphokinase (J. Melnick, E. Lis, J.-H. Park, H. Mori, C. Kinsland, J. Perkins, G. Schyns, A. Osterman, and T. P. Begley, submitted for publication). The pyrimidine and thiazole components can also be salvaged: thiazole is phosphorylated by thiazole kinase (2,4,6), HMP is phosphorylated to HMP-P by both ThiD and PdxK (3,7,10), and the phosphorylation of HMP-P is catalyzed by ThiD (5, 6, 7). Thus, ThiD has both a biosynthetic and a salvage function in thiamine biosynthesis.…”

Thiamine pyrophosphate (TPP) negatively regulates transcription of some thi genes of Salmonella typhimurium

“…The thiazole moiety is thought to be derived from cysteine, tyrosine, and 1-deoxy-Dthreo-2-pentulose (10, 12, 17), while the pyrimidine moiety (HMP) is thought to be derived from aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis (16, 18) and a proposed intermediate in the APB pathway (13). In E. coli, five kinases involved in TPP formation have been identified, and their genes have been mapped; thiL (min 10) encodes TMP kinase (22); thiK (min 25) encodes thiamine kinase (22); thiM, thiN, and thiD (min 46) encode THZ kinase, HMP kinase, and HMP-P kinase, respectively (22,27,28). Clearly, in E. coli and S. typhimurium, the genetic loci involved in thiamine synthesis are map throughout the chromosome (2, 31).…”

The thij locus and its relation to phosphorylation of hydroxymethylpyrimidine in Escherichia coli

“…0002-0837 0 1996 SGM close together on the chromosome at about 46 min (Mizote & Nakayama, 1989b). However, genetic analysis of HMP kinase (EC 2.7.1.49), which catalyses the phosphorylation of HMP to HMP-P, has not been possible because strains deficient in this enzyme have never been reported.…”

“…One such mutant, strain SN372L-3-9-10, was used for further studies. To determine whether or not the transductants had received the tbiM mutation, HET kinase activity was assayed in cell extracts by the procedure described previously (Mizote & Nakayama, 1989b). The results showed that 31 of 39 tbij' transductants possessed only about 1 /20 to 1 /40 the HET kinase activity of strain SN372L-3-9-10, while in the remaining 8 thiJ'transductants the kinase activities were comparable to those of the recipient, showing that the thi] mutation is 78-80 % linked to the tbiM allele.…”