The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

(5), compound 4 is the biosynthetic precursor of tetrahydrobiopterin, which is involved in the formation of catecholamines and nitric oxide (6, 7). Tetrahydrobiopterin is also involved in the stimulation of T lymphocytes, although the details are still incompletely understood (8).CYH was originally detected in bacteria (9). More recently, the primary structure of CYH from a wide variety of organisms has been determined (10-16). The evolution of the protein has been relatively conservative. Thus, the C-terminal 120 residues of Escherichia coli and human enzymes are 60% identical. The crystal structure of E. coli CYH has recently been solved by single isomorphous replacement and averaging techniquesThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (17). The knowledge of the crystal packing arrangement obtained by electron microscopy (18, 19) was helpful in the initial stages of structure determination. The enzyme complex, a decamer consisting of a pentamer of tightly associated dimers, is doughnut shaped with dimensions of 65 x 100 A.One CYH subunit folds into a a+f structure with a predominantly helical N terminus (Fig. 2). The N-terminal helix hl (residues 5-18) is followed by a helix pair, composed of helix h2 (residues 32-49) and helix h3 (residues 62-72), which are remote from the compact C-terminal domain of the molecule (residues 95-217). This domain has a central fourstranded antiparallel 13-sheet that is flanked on both sides by a-helices. The folding topology of this domain is identical to that of a subunit of the second enzyme in tetrahydrobiopterin biosynthesis pathway, 6-pyruvoyltetrahydropterin synthase (20).The association of two CYH monomers to dimers is driven by the formation of a four-helix bundle by helices h2 and h3 of each monomer. Apart from a large hydrophobic surface buried by this interaction, numerous hydrogen bonds and salt bridges serve to stabilize the dimer. The decamer is formed by a fivefold symmetric arrangement of the dimers. Its most striking structural feature is an unprecedented 20-stranded antiparallel ,B-barrel (Fig. 2). This paper describes the crystal structure of a complex of CYH with dGTP, the substrate analog.** By using the inforAbbreviation: CYH, GTP cyclohydrolase I.

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The ribG gene at the 5 end of the riboflavin operon of Bacillus subtilis and a reading frame at 442 kb on the Escherichia coli chromosome (subsequently designated ribD) show similarity with deoxycytidylate deaminase and with the RIB7 gene of Saccharomyces cerevisiae. The ribG gene of B. subtilis and the ribD gene of E. coli were expressed in recombinant E. coli strains and were shown to code for bifunctional proteins catalyzing the second and third steps in the biosynthesis of riboflavin, i.e., the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5-phosphate (deaminase) and the subsequent reduction of the ribosyl side chain (reductase). The recombinant proteins specified by the ribD gene of E. coli and the ribG gene of B. subtilis were purified to homogeneity. NADH as well as NADPH can be used as a cosubstrate for the reductase of both microorganisms under study. Expression of the N-terminal or C-terminal part of the RibG protein yielded proteins with deaminase or reductase activity, respectively; however, the truncated proteins were rather unstable.

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