Biosynthesis of the Thiazole Moiety of Thiamin Pyrophosphate (Vitamin B1) † Joo-Heon Park, Pieter C. Dorrestein, Huili Zhai, Cynthia Kinsland, Fred W. McLafferty, and Tadhg P. Begley* Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14853 ReceiVed May 28, 2003; ReVised Manuscript ReceiVed August 8, 2003 ABSTRACT: While most of the proteins required for the biosynthesis of thiamin pyrophosphate have been known for more than a decade, the reconstitution of this biosynthesis in a defined biochemical system has been difficult due to the novelty of the chemistry involved. Here we demonstrate the first successful enzymatic synthesis of the thiazole moiety of thiamin from glycine, cysteine, and deoxy-D-xylulose-5- phosphate using overexpressed Bacillus subtilis ThiF, ThiS, ThiO, ThiG, and a NifS-like protein. This has facilitated the identification of the biochemical function of each of the proteins involved: ThiF catalyzes the adenylation of ThiS; NifS catalyzes the transfer of sulfur from cysteine to the acyl adenylate of ThiS; ThiO catalyzes the oxidation of glycine to the corresponding imine; and ThiG catalyzes the formation of the thiazole phosphate ring. The complex oxidative cyclization reaction involved in the biosynthesis of the thiamin thiazole has been greatly simplified by replacing ThiF, ThiS, ThiO, and NifS with defined biosynthetic intermediates in a reaction where ThiG is the only required enzyme. Thiamin pyrophosphate is an essential cofactor in all living systems where it functions to stabilize the acyl carbanion synthon (1). The biosynthesis of this cofactor in Escherichia coli is outlined in Figure 1. The 4-amino-5-hydroxymethyl- 2-methylpyrimidine pyrophosphate (HMP-PP) 1 is formed from 5-aminoimidazole ribonucleotide (AIR) in a reaction involving a complex, poorly understood rearrangement; the 4-methyl-5-(-hydroxyethyl) thiazole phosphate (Thz-P) is formed from tyrosine, 1-deoxy-D-xylulose-5-phosphate (DXP), and cysteine; the thiazole and the pyrimidine are then coupled to give thiamin monophosphate (TMP), and a final phos- phorylation gives thiamin pyrophosphate (TPP), the biologi- cally active form of the cofactor (2-5). The formation of the thiazole moiety of thiamin is complex and requires six gene products: ThiS, ThiF, ThiG, ThiH (6), IscS (7), and ThiI (8, 9). The early steps in the sulfur incorporation chemistry have been worked out, and a novel acyl disulfide intermediate, covalently linking ThiF and ThiS, has been identified (10). However, despite the identification of an advanced intermediate, we have not been successful in reconstituting the thiazole biosynthesis using the E. coli proteins, possibly due to problems overexpressing and isolating active ThiH, a putative iron sulfur cluster containing protein. This analysis was recently supported by the isolation of ThiH under anaerobic conditions (11). In contrast to E. coli, Bacillus subtilis and many other bacteria do not use tyrosine or ThiH for the thiazole biosynthesis. Rather, these bacteria use glycine and ThiO, a stable flavoenzyme previously identified as a glycine oxidase (12-14). In this paper, we describe the successful reconstitu- tion of thiazole biosynthesis in a defined biochemical system containing ThiO, ThiF, ThiS, ThiG, and a cysteine des- ulfurase, and the identification of the function of each of these proteins. MATERIALS AND METHODS [ 35 S]-cysteine (20-150 mCi/mmol) was from Amersham Biosciences, [1,2- 13 C]-glycine was from Cambridge Isotope Laboratories (99% purity), and [1,2- 14 C]-glycine (101 mCi/ mmol) was from Amersham Biosciences. Thz-P, HMP, DXP, and [1- 13 C]-DXP were synthesized as previously described (15-17). Cloning of B. subtilis Thiazole Biosynthesis Genes. Standard methods were used for DNA restriction endonu- clease digestion, ligation, and transformation (18). Plasmid DNA was purified with the Wizard Plus SV DNA miniprep kit (Promega). DNA fragments were separated by agarose gel electrophoresis, excised, and purified with the QiaQuick Gel Extraction kit (Qiagen). The plasmid pET-16b was obtained from Novagen. E. coli strain DH5R was used as a recipient for transformation during plasmid construction and for plasmid propagation and storage. A Perkin-Elmer Ge- neAmp PCR system 2400 and Platinum Pfx DNA poly- merase (Gibco Life Technologies) were used for PCR. B. subtilis CU1065 genomic DNA was used as the template for PCR. Primer synthesis and DNA sequencing were carried out at the Cornell University Bioresource Center. Table 1 shows the plasmids constructed and used in this study. † This research was supported by grants from NIH to T.P.B. (DK44083) and to F.W.M. (GM16609) and by a gift from Hoffmann- La Roche. * To whom correspondence should be addressed. Tadhg P. Begley, Department of Chemistry and Chemical Biology, 120 Baker Laboratory, Cornell University, Ithaca, New York 14853. Phone: 607-255-7133. FAX: 607-255-4137. E-mail: tpb2@cornell.edu. 1 Abbreviations: AIR: 5-aminoimidazole ribonucleotide; DXP: 1-deoxy-D-xylulose-5-phosphate (DXP); ESI-FTMS: electrospray ion- ization-Fourier transform mass spectrometry; HMP: 4-amino-5-hy- droxymethyl-2-methylpyrimidine; HMP-P: 4-amino-5-hydroxymethyl- 2-methylpyrimidine phosphate; HMP-PP: 4-amino-5-hydroxymethyl- 2-methylpyrimidine pyrophosphate; IPTG: isopropyl--D-thiogalacto- pyranoside; PLP: pyridoxal phosphate; TCA: trichloroacetic acid; ThiS-COAMP: the carboxy terminal acyl adenylate of ThiS; ThiS- COSH: the carboxy terminal thiocarboxylate of ThiS; Thz-P: 4-methyl- 5-(-hydroxyethyl) thiazole phosphate; TMP: thiamin monophosphate; TPP: thiamin pyrophosphate. 12430 Biochemistry 2003, 42, 12430-12438 10.1021/bi034902z CCC: $25.00 © 2003 American Chemical Society Published on Web 09/30/2003