Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

Abstract

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-Å resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.Riboflavin (vitamin B 2 ) is biosynthesized in plants and numerous microorganisms but not in animals, which depend on nutritional sources. Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3, 4), and bioluminescence (5-8) and in reactions without net-redox change (9).Gram-negative bacteria are absolutely dependent on endogenous synthesis of riboflavin, because they are devoid of an uptake system for flavins or flavocoenzymes. Hence, riboflavindeficient mutants e.g. of Escherichia coli and Salmonella sp. require extremely high concentrations of exogenous riboflavin for growth. The same is true for yeasts such as Saccharomyces cerevisiae and Candida guilliermondii (for review see Refs. 1 and 10 -13). Thus, these organisms should be vulnerable to inhibitors of the riboflavin biosynthesis, which could therefore qualify as novel anti-infective agents. The absence of the riboflavin pathway in the human host appears advantageous in this context, because host/parasite selectivity of inhibitory agents would not constitute a problem. The mechanistic and structural analysis of the riboflavin pathway could serve as the basis for the rational design of riboflavin pathway inhibitors.3,4-Dihydroxy-2-butanone 4-phosphate synthase supplies the building blocks for the assembly of the xylene ring of the vitamin (14 -17). In fact, all eight carbon atoms of the xylene moiety are derived from the product of the enzyme. In the biosynthetic pathway, 3,4-dihydroxy-2-butanone 4-phosphate is condensed with 5-amino-6-ribitylamino-2,4(1H,3H)-...

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy- d -Xylulose 5-Phosphate

“…Nuclear magnetic resonance (NMR) and crystal structures have been solved for RibB from E. coli and other species, and a reaction mechanism has been proposed for conversion of Ru5P to the riboflavin precursor DHBP (39)(40)(41)(42). Several of the RibB mutations that enable production of DXP are at or close to catalytically important residues; for example, G108 is conserved among RibB proteins from diverse sources and is suggested by NMR studies to play a role in substrate binding (40), while crystal structure analysis suggests that the presence of a nonglycine residue at this critical turn would result in a sterically strained conformation (41).…”

Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase from Vibrio cholerae

“…The DHBPS structure complexed with substrate and metal ions indicates that an acidic active site loop (Loop1) and another loop (Loop2) undergo a conformational change upon substrate and/or metal binding (32,33,35). Furthermore, these structures also reveal the amino acids involved in the proposed reaction mechanism of DHBPS (27,28,(32)(33)(34)(35)(36). Nevertheless, certain differences observed in the existing structures prevent us from predicting the molecular mechanism of DHBPS completely.…”

“…In most of Gram-negative bacteria, DHBPS exists as monofunctional form, whereas in Gram-positive bacteria including Mycobacterium tuberculosis DHBPS co-exists with GTP cyclohydrolase II and thus is found in a bifunctional form (9,16,(27)(28)(29). Crystal structures of DHBPS from E. coli (30,31), Magnaporthe grisea (32), Methanococcus jannaschii (33), Candida albicans (34), Salmonella typhimurium (35), M. tuberculosis (28,36), and Streptococcus pneumoniae (27) have been reported. All these structures reveal that DHBPS is a homodimer where each monomer forms an ␣ ϩ ␤ fold and that its active site is located at two topologically equivalent positions at the dimeric interface of each monomer (27, 28, 30 -36).…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…The active site of 3,4-dihydroxy-2-butanone 4-phosphate synthase was localized by crystallographic analysis of the enzymes from the archaeon M. jannaschii and the pathogenic yeast Candida albicans in a complex with ribulose-5-phosphate (99,465,466). A highly conserved loop comprised of several acidic amino acid residues is essential for catalysis, as shown by studies with a variety of mutant proteins (120).…”

“…GTP cyclohydrolase is located at the C-terminal end of the fused protein, whereas 3,4-dihydroxy-2-butanone 4-phosphate synthase occupies the N-terminal part of the protein. The structure of this synthase was studied by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy (99,220,263,264,465,466). In addition to its known function in RF synthesis, the synthase also functions somehow in the regulation of mitochondrial respiration, as the corresponding S. cerevisiae rib3 knockout mutant grew with RF in a glucose medium but not in a glycerol or ethanol medium (197).…”