Nicotinamide Adenine Dinucleotide-dependent Proline Dehydrogenase in Chlorella

An NAD-linked dehydrogenase from Chlorella pyrenoidosa Chick catalyzing the conversion of l-proline to Delta(1)-pyrroline-5-carboxylic acid was partially purified. Delta(1)-Pyrroline-5-carboxylic acid was identified as the product by co-chromatography of it and its o-aminobenzaldehyde derivative with authentic compounds. The enzyme is NAD and l-proline specific and is not an oxidase; NADP is not inhibitory. The Michaelis constant for NAD is 0.08 mm and for proline is 0.73 mm.     

A radioisotopic assay for delta 1-pyrroline-5-carboxylate synthase activity

A radioisotopic assay is described for measuring the activity of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the formation of delta 1-pyrroline-5-carboxylic acid from glutamic acid. Pyrroline-5-carboxylic acid is a common intermediate in the pathways through which glutamic acid, proline, and ornithine are interconverted. To determine pyrroline-5-carboxylate synthase activity, cell homogenates are incubated with [14C]-glutamic acid, the products of the reaction are converted quantitatively to proline by sodium borohydride, and proline is isolated by cation-exchange column chromatography. Cofactor requirements have been defined, and the activity of pyrroline-5-carboxylate synthase in several different cultured fibroblast lines is reported.     

Fungal metabolism of biphenyl.

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.     

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.     

Purification and characterization of Delta(1)-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinacia oleracea L.) leaves

Delta(1)-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. L-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pathway, was purified from spinach (Spinacia oleracea L.) leaves. Following ammonium sulfate fractionation, purification was performed by several chromatographic methods: Blue Cellulofine, DEAE-TOYOPEARL, Sephacryl S-300 HR, and POROS QE/M. Two isoenzymes resolved by anion exchange chromatography were designated P5CR-1 and P5CR-2. Only P5CR-2 was purified from the intact chloroplasts, indicating differential distribution of the isoenzymes. P5CR isoenzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular mass of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa. P5CR-1 and P5CR-2 showed K(m) values of 9 and 19 microM for NADPH and values of 0.122 and 0.162 mM for Delta(1)-pyrroline-5-carboxylate (P5C), respectively. We decided partial amino acid sequences of P5CR-1 which showed the 70 to 80% homology to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibited by free ATP and Mg(2+) ion. The inhibition was partially mitigated when ATP and Mg(2+) were added simultaneously to the reaction mixture. Cations at high concentration were inhibitory to P5CR activity. Interestingly, P5CR-2 was more stable to heat treatment at 40 degrees C than P5CR-1.     

The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline

A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.     

Effect of the proline analogue baikiain on proline metabolism in Salmonella typhimurium

A proline analogue, 4,5-dehydro-l-pipecolic acid (baikiain) induces the formation in Salmonella typhimurium of the two enzymes catalyzing the degradation of proline, proline oxidase and Delta(1)-pyrroline-5-carboxylic acid (P5C) dehydrogenase. The level of induction by 20 mm baikiain is about 10% of the maximum level induced by proline. Since the analogue is a substrate of proline oxidase the first enzyme of the proline catabolic pathway, the oxidation derivative rather than baikiain itself might be the actual effector. Baikiain is also an inducer of proline oxidase in Escherichia coli K-12 and E. coli W. An additional effect of this analogue on proline degradation in S. typhimurium is inhibition of P5C dehydrogenase. At a concentration of 5 x 10(-4)m, baikiain inhibits completely the growth of strains constitutive for proline oxidase. This inhibition, which can be overcome by proline, occurs in the presence or absence of P5C dehydrogenase activity. Three spontaneously occurring mutants resistant to baikiain were isolated from constitutive strains. All are pleiotropic-negative for the proline-degrading enzymes. The sites of these mutations are linked to the put region. Although the mechanism of toxicity has not been determined, baikiain provides a simple and direct selection for obtaining mutants unable to degrade proline. In addition, it allows selection for strains with an inducible rather than constitutive phenotype.     

Enzymes metabolizing delta1-pyrroline-5-carboxylate in rat tissues.

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.     

Arginine and ornithine oxidation catalyzed by lentil seedling copper-amine oxidase

The oxidation of L-ornithine and L-arginine catalyzed by lentil (Lens esculenta) seedling copper-amine oxidase has been investigated by polarographic techniques, optical spectroscopy, and capillary electrophoresis. Both L-ornithine and L-arginine were found to be poor substrates for lentil amine oxidase. L-Ornithine was oxidized to glutamate-5-semialdehyde and ammonia, in similar manner as usual substrates. Glutamate-5-semialdehyde spontaneously cyclizes to ¹-pyrroline-5-carboxylic acid. Arginine is oxidized by an unusual mechanism yielding glutamate-5-semialdehyde, ammonia, and urea as reaction products.     

Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae

We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.     

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.     

Aldehyde oxidation in human placenta. Purification and properties of 1-pyrroline-5-carboxylate dehydrogenase.

The human placenta contains a considerable amount of 1-pyrroline-5-carboxylate dehydrogenase (23 +/- 6 micrograms/g; n = 12), about 25% of the concentration present in liver. The enzyme is the only form in placenta that oxidizes short- and medium-chain aldehydes, which facilitates its purification from this organ. It can be purified to homogeneity by successive chromatographies on DEAE-cellulose, 5'-AMP-Sepharose and Sephacryl S-300. From 500 g of tissue, about 2.1 units of enzyme can be obtained with a 12% yield. Placental 1-pyrroline-5-carboxylate dehydrogenase is a dimer of Mr-63,000 subunits. It exhibits a pI of 6.80-6.65, and is specific for 1-pyrroline-5-carboxylate, the cyclic form of glutamate gamma-semialdehyde (Km = 0.17 mM, kcat. = 870 min-1), although it also oxidizes short-chain aliphatic aldehydes such as propionaldehyde (Km = 24 mM, kcat. = 500 min-1). These properties are very close to those of the liver enzyme, indicating a strong similarity between the enzyme forms from both organs. The enzyme is highly sensitive to temperature, showing 50% inhibition after incubation for 0.8 min at 45 degrees C or after 23 min at 25 degrees C. It is irreversibly inhibited by disulfiram, and a molar ratio inhibitor: enzyme of 60:1 produced 50% inhibition after incubation for 10 min. A subcellular-distribution study indicates that the enzyme is located in two compartments: the mitochondria, with 60% of the total activity, and the cytosol, with 40% activity. The physiological role of the enzyme in placental amino acid metabolism is discussed.     

Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver

Delta1-pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyzes the conversion of Delta1-pyrroline-5-carboxylate to glutamate in a reaction requiring NADP+ as a cofactor. Delta1-pyrroline-5-carboxylate is formed in liver from proline by proline oxidase (EC number not assigned) or from ornithine via ornithine aminotransferase. A spectrophotometric assay for P5CDh was shown to be valid if rotenone was included in the assay to prevent reoxidation of NADH. Using this new assay, liver was fractionated using differential centrifugation and the distribution of P5CDh was compared to that of appropriate marker enzymes. P5CDh is enriched only in the mitochondrial fractions, as are the mitochondrial enzymes, succinate cytochrome c reductase, proline oxidase, glutaminase, and ornithine aminotransferase. Thus, it can be concluded that P5CDh occurs only in mitochondria, not in both mitochondria and cytoplasm, as had previously been reported.     

Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase

Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase     

Identity of proline dehydrogenase and delta1-pyrroline-5-carboxylic acid reductase in Clostridium sporogenes.

Proline dehydrogenase and delta1-pyrroline-5-carboxylic acid (PCA) reductase activities were copurified 60- and 130-fold, respectively, from extracts of Clostridium sporogenes. The primary change in the ratio of activites was the result of a loss of proline dehydrogenase activity during dialysis. Both activities were eluted in single peaks from diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200 columns. They had identical sedimentation coefficients (10.3S), as determined in linear sucrose gradients, and identical isoelectric points (4.95 to 5.12) based on isoelectric focusing. The proline dehydrogenase activity was dependent on nicotinamide adenine dinucleotide and L-proline, and the PCA reductase required L-PCA and reduced nicotinamide adenine dinucleotide. The optimum pH for the assay of proline dehydrogenase was approximately 10.2, whereas that for PCA reductase was 6.5 to 7.5. An increase in pH from 8.0 to 10.2 greatly decreased the apparent Michaelis constant observed for L-proline, and an increase from pH 8.3 to 8.6 resulted in a large shift in the reaction equilibrium toward PCA. Both the dehydrogenase and reductase activities were stabilized to heating at 65 degrees C for 5 min by solutes of high ionic strength and were inactivated in a similar fashion when dissolved in low-ionic-strength buffer. The specific activities for both were reduced by about 50% when glucose was added to the growth medium. The data support the conclusion that L-proline and L-PCA are interconverted by either a single enzyme or an enzyme complex in extracts of C. sporogenes cells.     

Purification, characterization, and application of a novel dye-linked L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

The distribution of dye-linked L-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked L-proline dehydrogenase (dye-L-proDH, L-proline:acceptor oxidoreductase) was found in the crude extract of some Thermococcales strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon, Thermococcus profundus DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (alpha(2) beta(2)) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the alpha-subunit (50-kDa subunit) and the beta-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-L-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70 degrees C for 10 min, and ca. 40% of the activity still remained after heating at 80 degrees C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50 degrees C for 10 min. The enzyme exclusively catalyzed L-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for L-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Delta(1)-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of L-proline at concentrations from 0.10 to 2.5 mM using the stable dye-L-proDH was achieved.     

Crystal structure of Thermus thermophilus Delta1-pyrroline-5-carboxylate dehydrogenase

Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDh) plays an important role in the metabolic pathway from proline to glutamate. It irreversibly catalyzes the oxidation of glutamate-gamma-semialdehyde, the product of the non-enzymatic hydrolysis of Delta(1)-pyrroline-5-carboxylate, into glutamate with the reduction of NAD(+) into NADH. We have confirmed the P5CDh activity of the Thermus thermophilus protein TT0033 (TtP5CDh), and determined the crystal structure of the enzyme in the ligand-free form at 1.4 A resolution. To investigate the structural basis of TtP5CDh function, the TtP5CDh structures with NAD(+), with NADH, and with its product glutamate were determined at 1.8 A, 1.9 A, and 1.4 A resolution, respectively. The solved structures suggest an overall view of the P5CDh catalytic mechanism and provide insights into the P5CDh deficiencies in the case of the human type II hyperprolinemia.     

Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum

Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.     

Pyridoxal phosphate de-activation by pyrroline-5-carboxylic acid. Increased risk of vitamin B6 deficiency and seizures in hyperprolinemia type II

We previously identified vitamin B6 deficiency in a child presenting with seizures whose primary diagnosis was the inherited disorder hyperprolinemia type II. This is an unrecognized association, which was not explained by diet or medication. We hypothesized that pyridoxal phosphate (vitamin B6 coenzyme) was de-activated by L-Delta(1)-pyrroline-5-carboxylic acid, the major intermediate that accumulates endogenously in hyperprolinemia type II. The proposed interaction has now been investigated in vitro with high resolution 1H nuclear magnetic resonance spectroscopy and mass spectrometry at a pH of 7.4 and temperature of 310 K. Three novel adducts were identified. These were the result of a Claisen condensation (or Knoevenagel type of reaction) of the activated C-4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. The structures of the adducts were confirmed by a combination of high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. This interaction has not been reported before. From preliminary observations, pyrroline-5-carboxylic acid also condenses with other aromatic and aliphatic aldehydes and ketones, and this may be a previously unsuspected generic addition reaction. Pyrroline-5-carboxylic acid is thus found to be a unique endogenous vitamin antagonist. Vitamin B6 de-activation may contribute to seizures in hyperprolinemia type II, which are so far unexplained, but they may be preventable with long term vitamin B6 supplementation.