Biosynthesis of riboflavin: characterization of the bifunctional deaminase-reductase of Escherichia coli and Bacillus subtilis.

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.     

Proline biosynthesis in Escherichia coli. Kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP+- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The Km for DL-pyrroline-5-carboxylic acid is 2.5 mM, for NADP+ is 0.05 mM and for phosphate is 0.35 mM. The Vmax is approx. 8.0 units per mg protein. The reaction is highly specific for the DL-pyrroline-5-carboxylic acid and NADP+, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of gamma-glutamyl phosphate, 3-(phosphonoacetylamido)-L-alanine, is competitive with respect to DL-pyrroline-5-carboxylic acid and non-competitive with respect to NADP+ and phosphate, suggesting dead-end complex formation.     

Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.     

Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-A resolution displayed a tetrameric ring-like structure with an extensive interface of approximately 2400 A(2)/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.     

3 linkage groups of the genes of riboflavin biosynthesis in Escherichia coli

Using transposon Tn5 inactivation technology a collection of Escherichia coli mutants defective in riboflavine biosynthesis was obtained. All mutations were distributed within three linkage groups. With the help of P1-transduction mapping, group I mutations (ribA locus) were localized near cysB locus (28 min of the standard 100 min E. coli map) and mutations of group II (ribB locus) were mapped near tolC locus (66 min). The location of group III mutations was approximately determined by the F' complementation analysis: this linkage group lies in the region of 56-60 min of the E. coli map.     

Proline biosynthesis in Escherichia coli kinetic and mechanistic properties of glutamate semialdehyde dehydrogenase

The kinetics of the NADP⁺- and phosphate-dependent oxidation of glutamic acid 5-semialdehyde are consistent with a rapid-equilibrium random order mechanism. The K_m for dl-pyrroline-5-carboxylic acid is 2.5 mM, for NADP⁺ is 0.05 mM and for phosphate is 0.35 mM. TheV_max is approx. 8.0 units per mg protein. The reaction is highly specific for the dl-pyrroline-5-carboxylic acid and NADP⁺, but a number of divalent anions can substitute for phosphate. NADPH is competitive with respect to all three substrates and an analog of γ-glutamyl phosphate, 3-(phosphonoacetylamido)-l-alanine, is competitive with respect to dl-pyrroline-5-carboxylic acid and non-competitive with respect to NADP⁺ and phosphate, suggesting dead-end complex formation.     

Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate in the pantothenate/coenzyme A biosynthetic pathway. The enzyme encoded by the panE gene from E. coli K12 was overexpressed and purified to homogeneity. The native enzyme exists in solution as a monomer with a molecular mass of 34 000 Da. The steady-state initial velocity and product inhibition patterns are consistent with an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding, and pantoate release precedes NADP(+) release. The pH dependence of the kinetic parameters V and V/K for substrates in both the forward and reverse reactions suggests the involvement of a single general acid/base in the catalytic mechanism. An enzyme group exhibiting a pK value of 8.4 +/- 0.2 functions as a general acid in the direction of the ketopantoate reduction, while an enzyme group exhibiting a pK value of 7.8 +/- 0.2 serves as a general base in the direction of pantoate oxidation. The stereospecific transfer of the pro-S hydrogen atom of NADPH to the C-2 position of ketopantoate was demonstrated by (1)H NMR spectroscopy. Primary deuterium kinetic isotope effects of 1.3 and 1.5 on V(for) and V/K(NADPH), respectively, and 2.1 and 1.3 on V(rev) and V/K(HP), respectively, suggest that hydride transfer is not rate-limiting in catalysis. Solvent kinetic isotope effects of 1.3 on both V(for) and V/K(KP), and 1.4 and 1.5 on V(rev) and V/K(HP), respectively, support this conclusion. The apparent equilibrium constant, K(eq)', of 676 at pH 7.5 and the standard free energy change, DeltaG, of -14 kcal/mol suggest that ketopantoate reductase reaction is very favorable in the physiologically important direction of pantoate formation.     

FtsK is a bifunctional protein involved in cell division and chromosome localization in Escherichia coli

The behaviour of Escherichia coli cells in which all or part of the ftsK gene is under inducible control shows that FtsK protein has two functional domains: an N-terminal part that is required for cell division, and a C-terminal part that is involved in chromosome localization within the cell.     

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.     

Cloning and characterization of genes involved in the biosynthesis of delta-aminolevulinic acid in Escherichia coli.

Several mutants of Escherichia coli that had lost their ability to synthesize delta-aminolevulinic acid (ALA) via the C5 pathway were isolated. Their defective loci were classified into two groups, AlaA- and AlaB-. The genes that complemented these mutations were cloned. Nucleotide sequencing indicated that the gene that complemented AlaA- was identical to hemL which is located at 4 min on the E. coli chromosome and encodes glutamate 1-semialdehyde aminotransferase. The gene complementing AlaB- contained an open reading frame (ORF) encoding a polypeptide of 207 amino acids that was found to be a new gene involved in the synthesis of ALA via the C5 pathway. Thus, we designated the gene hemM. The hemM gene was adjacent to hemA that is located at 27 min and previously thought to encode glutamyl-tRNA dehydrogenase. However, we found that hemA complemented both the AlaA- (hemL) and AlaB- (hemM) mutants defective in the C5 pathway although the transformants showed small colonies on the selective medium without ALA. These results suggest that hemA is not involved in the C5 pathway, but controls a second, minor pathway for the synthesis of ALA.     

Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis.

gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.     

Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase

A kinetic mechanism is presented for Escherichia coli dihydrofolate reductase which describes the full time course of the enzymatic reaction over a wide range of substrate and enzyme concentrations at pH 7.2 and 20 degrees C. Specific rate constants were estimated by computer simulation of the full time course of single turnover, burst, and steady-state experiments using both nondeuterated and deuterated NADPH. The mechanism involves the random addition of substrates, but the substrates and enzyme are not at equilibrium prior to the chemical transformation step. The rate-limiting step follows the chemical transformation, and the maximum velocity of the reaction is limited by the release of the product tetrahydrofolate. The full time course of the reaction is markedly affected by the formation of the enzyme-NADPH-tetrahydrofolate abortive complex, but not by the enzyme-NADP-dihydrofolate abortive complex.     

Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g _CDW ^?1 h^?1 and 0.0035 mM s^?1, respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).     

Enzyme biosynthesis in Escherichia coli

Escherichia coli B synthesized beta-galactosidase and an enzyme system for D-xylose when exposed to lactose and xylose respectively in nitrogen-free media. The amount of beta-galactosidase formed in the absence of external nitrogen depended upon the nature of the medium in which the cells had originally been grown. Half as much of this enzyme was synthesized without exogenous nitrogen by cells taken from a nitrogen-rich medium as was formed by cells under favorable conditions with an external supply of nitrogen. Escherichia coli B contained a pool of nitrogen compounds soluble in 80 per cent ethanol and made up of several ninhydrin-positive components. One of these was identified chromatographically as glycine using an authentic radioactive sample. Another substance behaved like serine on the chromatograms. The internal pool of amino acids and peptides was large enough to account for the beta-galactosidase synthesized by cells exposed to lactose in a medium free of nitrogen. Some degree of interaction of the syntheses of the beta-galactosidase and xylose enzyme systems was observed in nitrogen-free media. This interaction produced a greater effect on the formation of beta-galactosidase and was attributed to a limiting factor(s) in the internal nitrogenous pool or to a limiting intermediate in enzyme synthesis.     

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.