title: Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities.
abstract: A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.
authors: Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. rapala@mol.uj.edu.pl
authors: Maria
authors: M
authors: Rapala-Kozik
authors: Mariusz
authors: M
authors: Olczak
authors: Katarzyna
authors: K
authors: Ostrowska
authors: Agata
authors: A
authors: Starosta
authors: Andrzej
authors: A
authors: Kozik
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013831:Thiamine / Q000096:biosynthesis* / Q000737:chemistry; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
chemical_list: C101901:4-amino-5-hydroxymethyl-2-methylpyrimidine; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D010940:Plant Proteins; D011743:Pyrimidines; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C435495:thiamin phosphate synthase; C108436:4-amino-5-(hydroxymethyl)-2-methylpyrimidine phosphate kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); D013831:Thiamine
references: Bioorg Chem. 2005 Aug;33(4):338-44
references: 15967475
references: Arch Microbiol. 1999 Apr;171(5):293-300
references: 10382260
references: Biochemistry. 2006 Sep 19;45(37):11061-70
references: 16964967
references: J Biol Chem. 2006 Oct 13;281(41):30957-66
references: 16912043
references: Structure. 2001 Jun;9(6):539-46
references: 11435118
references: Biochemistry. 2001 Aug 28;40(34):10103-14
references: 11513589
references: Structure. 2002 Feb;10(2):225-35
references: 11839308
references: Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
references: 12824332
references: Curr Opin Struct Biol. 2003 Dec;13(6):739-47
references: 14675553
references: Bioinformatics. 2004 Feb 12;20(3):426-7
references: 14960472
references: J Am Chem Soc. 2004 Mar 17;126(10):3091-6
references: 15012138
references: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W327-31
references: 15215404
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: Methods Enzymol. 1979;62:69-73
references: 374983
references: J Bacteriol. 1980 Jan;141(1):254-61
references: 6986359
references: Ann N Y Acad Sci. 1982;378:161-76
references: 6282163
references: J Chromatogr. 1991 Jun 14;567(1):71-80
references: 1918262
references: Methods Enzymol. 1993;218:340-56
references: 7685466
references: J Bacteriol. 1993 Aug;175(16):5153-8
references: 8394314
references: Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
references: 7984417
references: Plant Mol Biol. 1995 Nov;29(4):809-21
references: 8541506
references: Plant J. 1996 Aug;10(2):361-8
references: 8771789
references: Plant Mol Biol. 1996 Jun;31(3):585-93
references: 8790291
references: J Bacteriol. 1997 May;179(9):3030-5
references: 9139923
references: Methods Enzymol. 1997;279:109-17
references: 9211263
references: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references: 9254694
references: J Mol Biol. 1997 Oct 17;273(1):114-21
references: 9367751
references: J Biol Chem. 1998 Apr 24;273(17):10567-77
references: 9553117
references: Plant Mol Biol. 1998 Aug;37(6):955-66
references: 9700068
references: Protein Sci. 1999 May;8(5):978-84
references: 10338008
references: Biochemistry. 1999 May 18;38(20):6460-70
references: 10350464
references: Appl Microbiol Biotechnol. 2006 Aug;72(1):30-40
references: 16826377