Isoprenoid biosynthesis in plants - 2C-methyl-D-erythritol-4-phosphate synthase (IspC protein) of Arabidopsis thaliana.

The ispC gene of Arabidopsis thaliana was expressed in pseudomature form without the putative plastid-targeting sequence in a recombinant Escherichia coli strain. The recombinant protein was purified by affinity chromatography and was shown to catalyze the formation of 2C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate at a rate of 5.6 micromol x min(-1) x mg(-1) (k(cat) 4.4 s(-1)). The Michaelis constants for 1-deoxy-D-xylulose 5-phosphate and the cosubstrate NADPH are 132 and 30 microm, respectively. The enzyme has an absolute requirement for divalent metal ions, preferably Mn2+ and Mg2+, and is inhibited by fosmidomycin with a Ki of 85 nm. The pH optimum is 8.0. NADH can substitute for NADPH, albeit at a low rate (14% as compared to NADPH). The enzyme catalyzes the reverse reaction at a rate of 2.1 micromol x min(-1) x mg(-1).

Evolution of vitamin B2 biosynthesis: riboflavin synthase of Arabidopsis thaliana and its inhibition by riboflavin.

A synthetic gene specifying the catalytic domain of the Arabidopsis thaliana riboflavin synthase was expressed with high efficiency in a recombinant Escherichia coli strain. The recombinant pseudomature protein was shown to convert 6,7-dimethyl-8-ribityllumazine into riboflavin at a rate of 0.027 s-1 at 25 degrees C. The protein sediments at a rate of 3.9 S. Sedimentation equilibrium analysis afforded a molecular mass of 67.5 kDa, indicating a homotrimeric structure, analogous to the riboflavin synthases of Eubacteria and fungi. The protein binds its product riboflavin with relatively high affinity (Kd =1.1 microM). Product inhibition results in a characteristic sigmoidal velocity versus substrate concentration relationship. Characterization of the enzyme/product complex by circular dichroism and UV absorbance spectroscopy revealed a shift of the absorption maxima of riboflavin from 370 and 445 to 399 and 465 nm, respectively. Complete or partial sequences for riboflavin synthase orthologs were analyzed from 11 plant species. In each case for which the complete plant gene sequence was available, the catalytic domain was preceded by a sequence of 1-72 amino acid residues believed to function as plastid targeting signals. Comparison of all available riboflavin synthase sequences indicates that hypothetical gene duplication conducive to the two-domain architecture occurred very early in evolution.

Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments.

BACKGROUND: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs. RESULTS: A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20 degrees C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg(-1) min(-1) with an apparent KM of 5.7 microM. 19F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. 19F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins. CONCLUSION: Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The 19F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.

Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.

A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively. The enzyme binds riboflavin with a Kd of 1.2 microm. The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin. The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.