Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.     

Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Deltaprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2) required inorganic phosphate for activity, whereas the others were phosphate independent. PRPP synthase isozymes 2 and 3 contained 76 and 87 amino acid extensions, respectively, at their N-terminal ends in comparison with other PRPP synthases. Isozyme 2 was synthesized in vitro and shown to be imported and processed by pea (Pisum sativum) chloroplasts. Amino acid sequence analysis indicated that isozyme 3 may be transported to mitochondria and that isozyme 4 may be located in the cytosol. The deduced amino acid sequences of isozymes 1 and 2 and isozymes 3 and 4 were 88% and 75% identical, respectively. In contrast, the amino acid identities of PRPP synthase isozyme 1 or 2 with 3 or 4 was modest (22%-25%), but the sequence motifs for binding of PRPP and divalent cation-nucleotide were identified in all four sequences. The results indicate that PRPP synthase isozymes 3 and 4 belong to a new class of PRPP synthases that may be specific to plants.     

Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate. This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA. DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0. 77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.     

Enzymatic synthesis of diastereospecific carbacephem intermediates using serine hydroxymethyltransferase

The serine hydroxymethyltransferase (SHMT) gene glyA was over-expressed in Escherichia coli and the enzyme was purified to near homogeneity. Reaction conditions for E. coli and rabbit liver SHMTs were optimized using succinic semialdehyde methyl ester (SSAME) and glycine. The catalytic efficiency (kcat/K(m)) of E. coli SHMT for SSAME was 2.8-fold higher than that of rabbit liver enzyme. E. coli SHMT displayed a pH-dependent product distribution different from that of rabbit liver enzyme. For the pyridoxal-5'-phosphate (PLP)-dependent reaction, E. coli and rabbit liver SHMTs showed a high product diastereospecificity. The stoichiometric ratio of PLP to the dimeric E. coli SHMT was 0.5-0.7, indicating a requirement for external PLP for maximal activity. Using SSAME or its analog at a high temperature, E. coli SHMT mediated efficient condensation via a lactone pathway. In contrast, at a low temperature, the enzyme catalyzed efficient conversion of 4-penten-1-al via a non-lactone mechanism. Efficient conversion of either aldehyde type to a desirable diastereospecific product was observed at a pilot scale. E. coli SHMT exhibited a broad specificity toward aldehyde substrates; thus it can be broadly useful in chemo-enzymatic synthesis of a chiral intermediate in the manufacture of an important carbacephem antibiotic.     

Slow-binding and competitive inhibition of 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs. Kinetic and binding studies

8-Amino-7-oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro-organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8-amino-7-oxopelargonate synthase using six different compounds. d-Alanine, the enantiomer of the substrate of this pyridoxal-5'-phosphate-dependent enzyme was found to be a competitive inhibitor with respect to l-alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l-alanine was interpreted as the consequence of the inversion-retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d-alanine and pyridoxal-5'-phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d-alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d-alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl-CoA, the second substrate. This result strongly suggests that the binding of pimeloyl-CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d-alanine and to the putative reaction intermediate since they both have the same configuration. (+/-)-8-Amino-7-oxo-8-phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 microm. This compound behaved as a reversible slow-binding inhibitor, competitive with respect to l-alanine. Kinetic investigation showed that this slow process was best described by a one-step mechanism (mechanism A) with the following rate constants: k1 = 0.27 x 103 m-1.s-1, k2 = 1.8 s-1 and half-life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using ultraviolet/visible spectroscopy, and the data were consistent with the kinetic data (K = 4.2 microm). Among the other compounds tested, two potential transition state analogs, 4-carboxybutyl(1-amino-1-carboxyethyl)phosphonate (4) and 2-amino-3-hydroxy-2-methylnonadioic acid (5) were found to be competitive inhibitors with respect to l-alanine with Ki of 68 microm and 80 microm, respectively.     

Mechanistic studies on the 8-amino-7-oxopelargonate synthase, a pyridoxal-5'-phosphate-dependent enzyme involved in biotin biosynthesis

The reaction mechanism of 8-amino-7-oxopelargonate (8-amino-7-oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5'-phosphate (pyridoxal-P), which catalyzes the condensation of L-alanine with pimeloyl-CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over-expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8-amino-7-oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal-P-stabilized carbanion by abstraction of the C2-H proton of the alanine-pyridoxal-P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2-H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8-amino-7-oxopelargonate formed using either L-[2-2H]alanine in H2O or L-alanine in D2O, showed that the C2-H proton of alanine is lost during the reaction and that the C8-H proton of 8-amino-7-oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Furthermore 8-amino-7-oxopelargonate synthase catalyzes, in the absence of pimeloyl-CoA, the stereospecific exchange, with retention of configuration, of the C2-H proton of L-alanine with the solvent protons. Similarly, 8-amino-7-oxopelargonate synthase catalyzes the exchange of the C8-H proton of 8-amino-7-oxopelargonate. In addition to these exchange reactions, 8-amino-7-oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5'-phosphate (pyridoxamine-P) form of 8-amino-7-oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch. = 1.8 min-1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans. = 0.11 h-1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L-alanine (DV = 1.3) and in D2O (D2OV = 4.0). The magnitudes of the KIE are consistent with a partially rate-limiting abstraction of the C2-H proton of alanine and a partially rate-limiting reprotonation step. Taken together, all these results show that 8-amino-7-oxopelargonate synthase utilizes mechanism (a). 8-Amino-7-oxopelargonate synthase and 5-aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal-P-dependent enzymes that catalyze the formation of alpha-oxoamines. Based on the fact that all these alpha-oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism.     

Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase

The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from 18O2 and one atom of oxygen from H218O are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway of animals, fungi and some bacteria. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. In mouse erythroid 5-aminolevulinate synthase, lysine 313 has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., et al. (1993) Protein Sci. 2, 1959-1965], while arginine 149, a conserved residue among all known 5-aminolevulinate synthase sequences, is essential for function [Gong & Ferreira (1995) Biochemistry 34, 1678-1685]. To determine whether each subunit contains an independent active site (i.e., intrasubunit arrangement) or whether the active site resides at the subunit interface (i.e., intersubunit arrangement), in vivo complementation studies were used to generate heterodimers from site-directed, catalytically inactive mouse 5-aminolevulinate synthase mutants. When R149A and K313A mutants were co-expressed in a hem A- Escherichia coli strain, which can only grow in the presence of 5-aminolevulinate or when it is transformed with an active 5-aminolevulinate synthase expression plasmid, the hem A- E. coli strain acquired heme prototrophy. The purified K313A/R149A heterodimer mixture exhibited K(m) values for the substrates similar to those of the wild-type enzyme and approximately 26% of the wild-type enzyme activity which is in agreement with the expected 25% value for the K313A/R149A coexpression system. In addition, DNA sequencing of four Saccharomyces cerevisiae 5-aminolevulinate synthase mutants, which lack ALAS activity but exhibit enzymatic complementation, revealed that mutant G101 with mutations N157Y and N162S can complement mutant G220 with mutation T452R, and mutant G205 with mutation C145R can complement mutant Ole3 with mutation G344C. Taken together, these results provide conclusive evidence that the 5-aminolevulinate synthase active site is located at the subunit interface and contains catalytically essential residues from the two subunits.     

General acid-base catalysis of alpha-glucan phosphorylases: stereospecific glucosyl transfer from D-glucal is a pyridoxal 5'-phosphate and orthophosphate (arsenate) dependent reaction

D-Glucal, containing a highly reactive double bond, can replace glucose 1-phosphate as the glucosyl donor in phosphorylase-catalyzed glucosyl transfer to a suitable oligo- or polysaccharide acceptor: D-glucal + Pi + (glucose)Pi leads to n 2-deoxy-alpha-D-glucosyl(glucose)n in equilibrium 2-deoxy-alpha-D-glucose-1-P + (glucose)n. This reaction is catalyzed by alpha-glucan phosphorylases from rabbit skeletal muscle, potato tuber, and Escherichia coli. D-Glucal is only measurably consumed by alpha-glucan phosphorylases when orthophosphate or arsenate is present. With saturating concentrations of these anions and a glucosyl acceptor, the D-glucal reaction proceeds at rates comparable with the rates of glucosyl transfer from glucose 1-phosphate and of phosphorolysis or arsenolysis of poly- or oligosaccharides. Furthermore, for the reaction to proceed, the enzyme must be in the active conformation containing the cofactor pyridoxal 5'-phosphate in its dianionic form. On the basis of proton nuclear magnetic resonance spectra, it is proposed that protonation at C-2 of D-glucal gives rise to a hypothetical 2-deoxy-beta-D-glucose intermediate, yielding as a final product (2-deoxy-alpha-D-[2(e)-2H]glucose)n alpha (1 leads to 4) saccharides. These 2-deoxy-alpha-D-glucose oligo- or polysaccharides are degraded by alpha-glucan phosphorylases by phosphorolysis or arsenolysis like natural linear and branched alpha-glucans. The absolute requirement of the D-glucal reaction for phosphate (or arsenate) and its dependency on the dianionic form of the pyridoxal 5'-phosphate bound to phosphorylase are rationalized in terms of a proton transfer relay involving juxtaposed phosphates. Phosphate--phosphate interactions were postulated by Withers et al. [Withers, S. G., Madsen, N. B., Sykes, B. D., Takagi, M., Shimomura, S., & Fukui, T. (1981) J. Biol. Chem. 256, 10759-10762].     

Effect of NADPH-associated keto-reducing domain on substrate entry into 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme involved in phytoalexin biosynthesis in carrot

6-Hydroxymellein synthase is a polyketide biosynthetic enzyme induced in carrot cells which is organized as a homodimer composed of multifunctional subunits. The synthase liberates triacetic acid lactone, instead of 6-hydroxymellein, as a derailment product when the keto-reducing reaction at the triketide intermediate stage is interrupted. However, the efficiency of the triacetic acid lactone-forming reactions is markedly lower than that of the normal reaction, and the kinetic analyses have revealed that the affinity of the enzyme protein for acetyl-CoA is appreciably reduced in the abnormal reactions. It is assumed that the interaction of the NADPH-associated keto-reducing domain with a putative primary binding site(s) of the acyl-CoA in the enzyme structure affects the entry of the starter unit into the protein. The present finding should provide an example of the novel class of "subunit communication" of multimer enzymes.     

Proteinase K from the mold Tritirachium album Limber. Specificity and mode of action

1) The specificity of proteinase K towards amino acid and oligopeptide nitroanilide substrates is investigated. 2) The active center of the enzyme contains an extended binding region consisting of several subsites. An integral part of the S1-subsite are hydrophobic areas which were investigated by systematic elongation of the carbon skeleton in carboxylic acid 4-nitrophenyl esters. On the basis of these studies, a possible model of the S1-binding site is proposed. 3) Kinetic parameters for the hydrolysis of substituted phenyl acetates catalyzed by proteinase K have been measured at pH 7 and 25 degrees C. Deacylation of an acyl-enzyme intermediate is probably the rate-limiting step. Acylation (kcat/km used as a measure) is modestly sensitive to the sigma values of the substituents (p = 1.33, r = 0.9108), indicating electrophilic assistance by the enzyme in the catalytic mechanism. 4) Hydrophobic forces apparently are not involved in the binding of the leaving group.     

IL-6 expression by oral fibroblasts is regulated by androgen

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.     

Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (Part I)

Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Cornybacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY12505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.     

Effect of phospholipase C on lymphocyte responses to mitogen

A reinvestigation of flavin phosphate synthesis, separation, identification, and interconversion was made in view of contradictory results in the literature. It has been confirmed that monochlorophosphoric acid is the best agent for selective 5'-monophosphorylation of riboflavin and derivatives. This reaction yields, however, invariably up to 20% of an isomer, which has been separated by preparative thick-layer chromatography and shown to be the 4'-monophosphate. All the earlier authors failed to detect this isomer which does not bind to flavodoxin. It equilibrates in dilute mineral acid to yield an 8:2 mixture of 5'-phosphate to 4'-phosphate by phosphate migration. The formation of 2',3',4'-triacetyl-flavin mononucleotide, according to Christie, S. M. H., Kenner, G. W. & Todd, A. R. (1954) J. Chem. Soc., 46-52, upon acid-catalysed acetylation of pure FMN, was confirmed. The same reaction under base catalysis, however, does not yield 2',3',4'-triacetyl-flavin mononucleotide as claimed by Khomutova, E. D., Shapiro, T. A., Mezentseva, M. W. & Berezovskii, V. M. (1965) Otd. Obshch. i. Tekhn. Khim., 241-244, Chem. Abstr. 65, 5516a, but in fact up to 80% 2', 3'-diacetyl-flavin 4':5'-cyclophosphate as the main product, which is stable under neutral and weak acidic conditions and does not hydrolyse to 2',3'-diacetyl-flavin 5'-monophosphate as claimed by McCormick, D.B. (1974) J. Heterocycl. Chem. 11, 969-974. The various flavin phosphates and their acetyl derivatives have been identified by pH-titration, electrophoresis, and proton magnetic resonance spectrometry, which direct analyses of crude reaction products as well as a rapid purity check of commerical FMN.     

Role of arginine 439 in substrate binding of 5-aminolevulinate synthase

5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme. In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.     

Nucleon-nucleon pion-exchange tested in three-body reactions

The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.     

Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor

5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.     

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.     

Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 microM for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.