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.
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.
Rapala-Kozik
Maria
M
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. rapala@mol.uj.edu.pl
Olczak
Mariusz
M
Ostrowska
Katarzyna
K
Starosta
Agata
A
Kozik
Andrzej
A
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
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
Bioorg Chem. 2005 Aug;33(4):338-44
15967475
Arch Microbiol. 1999 Apr;171(5):293-300
10382260
Biochemistry. 2006 Sep 19;45(37):11061-70
16964967
J Biol Chem. 2006 Oct 13;281(41):30957-66
16912043
Structure. 2001 Jun;9(6):539-46
11435118
Biochemistry. 2001 Aug 28;40(34):10103-14
11513589
Structure. 2002 Feb;10(2):225-35
11839308
Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
12824332
Curr Opin Struct Biol. 2003 Dec;13(6):739-47
14675553
Bioinformatics. 2004 Feb 12;20(3):426-7
14960472
J Am Chem Soc. 2004 Mar 17;126(10):3091-6
15012138
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W327-31
15215404
Nature. 1970 Aug 15;227(5259):680-5
5432063
Methods Enzymol. 1979;62:69-73
374983
J Bacteriol. 1980 Jan;141(1):254-61
6986359
Ann N Y Acad Sci. 1982;378:161-76
6282163
J Chromatogr. 1991 Jun 14;567(1):71-80
1918262
Methods Enzymol. 1993;218:340-56
7685466
J Bacteriol. 1993 Aug;175(16):5153-8
8394314
Nucleic Acids Res. 1994 Nov 11;22(22):4673-80
7984417
Plant Mol Biol. 1995 Nov;29(4):809-21
8541506
Plant J. 1996 Aug;10(2):361-8
8771789
Plant Mol Biol. 1996 Jun;31(3):585-93
8790291
J Bacteriol. 1997 May;179(9):3030-5
9139923
Methods Enzymol. 1997;279:109-17
9211263
Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
9254694
J Mol Biol. 1997 Oct 17;273(1):114-21
9367751
J Biol Chem. 1998 Apr 24;273(17):10567-77
9553117
Plant Mol Biol. 1998 Aug;37(6):955-66
9700068
Protein Sci. 1999 May;8(5):978-84
10338008
Biochemistry. 1999 May 18;38(20):6460-70
10350464
Appl Microbiol Biotechnol. 2006 Aug;72(1):30-40
16826377