Biosynthesis of riboflavin: structure and properties of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase of Methanocaldococcus jannaschii.
The pyrimidine reductase of the riboflavin biosynthetic pathway (MjaRED) specified by the open reading frame MJ0671 of Methanocaldococcus jannaschii was expressed in Escherichia coli using a synthetic gene. The synthetic open reading frame that was optimized for expression in E. coli directed the synthesis of abundant amounts of the enzyme with an apparent subunit mass of 25 kDa. The enzyme was purified to apparent homogeneity and was shown to catalyze the conversion of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate at a rate of 0.8 micromol min(-1) mg(-1) at pH 8.0 and at 30 degrees C. The protein is a homodimer as shown by sedimentation equilibrium analysis and sediments at an apparent velocity of 3.5 S. The structure of the enzyme in complex with the cofactor nicotinamide adenine dinucleotide phosphate was determined by X-ray crystallography at a resolution of 2.5 Angstroms. The folding pattern resembles that of dihydrofolate reductase with the Thermotoga maritima ortholog as the most similar structure. The substrate, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, was modeled into the putative active site. The model suggests the transfer of the pro-R hydrogen of C-4 of NADPH to C-1' of the substrate.
Chatwell
Lorenz
L
Max-Planck Institut für Biochemie, Abteilung für Strukturforschung, Martinsried, Germany. chatwell@wzw.tum.de
Krojer
Tobias
T
Fidler
Alexander
A
Römisch
Werner
W
Eisenreich
Wolfgang
W
Bacher
Adelbert
A
Huber
Robert
R
Fischer
Markus
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D015870:Gene Expression; D005813:Genes, Synthetic / Q000235:genetics; D017015:Methanococcales / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012256:Riboflavin / Q000096:biosynthesis* / Q000737:chemistry; D016415:Sequence Alignment; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology
D003067:Coenzymes; D013401:Sugar Alcohol Dehydrogenases; C033549:2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase; D013762:Tetrahydrofolate Dehydrogenase; D012256:Riboflavin