Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana.

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The Vmax/Km values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.

Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation.

Lys49 phospholipase A2 (PLA2) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants map to the C-terminal region 115-129, which plays an effector role in membrane damage. The dimeric state was reported to be essential for a Lys49 PLA2 which lost its liposome-disrupting activity after dissociating into monomers at pH 5.0. This study, evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA2s, using biological targets instead. Both their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides of two Lys49 PLA2s also showed lower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the dimeric/monomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the dimeric state of Lys49 PLA2s could play an enhancing, although not essential role, in their C-terminal region-mediated mechanism of myotoxicity.

Synthesis and evaluation of mimetics of UDP and UDP-alpha-D-galactose, dTDP and dTDP-alpha-D-glucose with monosaccharides replacing the key pyrophosphate unit.

A series of 5'-O-glycosyl-uridine and thymidine derivatives have been prepared as potential mimics of sugar nucleotides and nucleotide-diphosphates. These compounds proved not to be inhibitors of bovine beta-1,4-galactosyltransferase although some showed moderate inhibition of Salmonella dTDP-alpha-D-glucose 4,6-dehydratase (RmlB).

Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus.

Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron-sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of Ti(III) citrate or dithionite as reducing agents.

NaoXinQing, an anti-stroke herbal medicine, reduces hydrogen peroxide-induced injury in NG108-15 cells.

NaoXinQing (NXQ) is a patented and approved drug of Traditional Chinese Medicine that has been used for years for the treatment of syndrome of apoplexy, but the underlying mechanism remains unclear. The present study is designed to investigate the effects of NXQ on hydrogen peroxide (H(2)O(2))-induced cell damage in NG108-15 cells. Exposure to H(2)O(2) induces apoptosis-like cell injury. Preincubation of cells with NXQ alleviated H(2)O(2)-induced cell injury and apoptosis. This herb medicine also improves redox imbalance in cells under the exposure of H(2)O(2) as indicated by the attenuation in the reduction of activities of intracellular endogenous antioxidants, glutathione and glutathione peroxidase as well as catalase, and by the decrease in the leak of lactate dehydrogenase and the accumulation of malondialdehyde. These results indicate that NXQ significantly protects NG108-15 cells against H(2)O(2) challenge by improving redox imbalance and inhibiting apoptosis, which might represent mechanisms underlying its potential usage in the prevention and treatment of syndrome of apoplexy.

Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants.

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.

Genomic profiling of drug sensitivities via induced haploinsufficiency.

Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analysed for drug sensitivity using high-density oligonucleotide arrays. Individual heterozygous strain analysis verified six known drug targets. Parallel analysis identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity observed in human populations.

Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells.

High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.

Highly efficient control of iron-containing nitrile hydratases by stoichiometric amounts of nitric oxide and light.

The reaction of two iron-containing nitrile hydratases (NHase) with NO has been studied: NHase from Rhodococcus sp. R312, which is probably similar to the photosensitive N771 NHase, and the new NHase from Comamonas testosteroni NI1 whose aminoacid sequence is quite different from those of BR312 and N771 NHases. Both enzymes are equally inactivated after addition of stoichiometric amounts of NO added as an anaerobic solution or produced in situ under physiological conditions by a rat brain NO-synthase. Both enzymes are reactivated by photoirradiation, and two cycles of NO inactivation/photoactivation can be performed without significant loss of activity. Both iron-containing NHases have a high affinity for NO, similar to that of methemoglobin.

Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase.

In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene. We previously cloned the soybean (Glycine max cv. Century) DapA gene in Escherichia coli to express functional soybean DS protein. Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine. Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications. When expressed in E. coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM.

Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein.

Acetylene hydratase of the mesophilic fermenting bacterium Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde. Growth of P. acetylenicus with acetylene and specific acetylene hydratase activity depended on tungstate or, to a lower degree, molybdate supply in the medium. The specific enzyme activity in cell extract was highest after growth in the presence of tungstate. Enzyme activity was stable even after prolonged storage of the cell extract or of the purified protein under air. However, enzyme activity could be measured only in the presence of a strong reducing agent such as titanium(III) citrate or dithionite. The enzyme was purified 240-fold by ammonium sulfate precipitation, anion-exchange chromatography, size exclusion chromatography, and a second anion-exchange chromatography step, with a yield of 36%. The protein was a monomer with an apparent molecular mass of 73 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was at pH 4.2. Per mol of enzyme, 4.8 mol of iron, 3.9 mol of acid-labile sulfur, and 0.4 mol of tungsten, but no molybdenum, were detected. The Km for acetylene as assayed in a coupled photometric test with yeast alcohol dehydrogenase and NADH was 14 microM, and the Vmax was 69 mumol.min-1.mg of protein-1. The optimum temperature for activity was 50 degrees C, and the apparent pH optimum was 6.0 to 6.5. The N-terminal amino acid sequence gave no indication of resemblance to any enzyme protein described so far.

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase.

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA, as a lacZ fusion protein was selected in a dapA- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.

Characterization of the type I dehydroquinase from Salmonella typhi.

The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity. The S. typhi enzyme was characterized in terms of its kinetic parameters, important active-site residues, thermal stability and c.d. and fluorescence properties. In all important respects, the enzyme from S. typhi behaves in a very similar fashion to the well-characterized enzyme from E. coli, including the remarkable conformational stabilization observed on reduction of the substrate/product mixture by NaBH4. This gives confidence that the information from X-ray studies on the S. typhi enzyme [Boys, Fawcett, Sawyer, Moore, Charles, Hawkins, Deka, Kleanthous and Coggins (1992) J. Mol. Biol. 227, 352-355] can be applied to other type I dehydroquinases. Studies of the quenching of fluorescence of the S. typhi enzyme by succinimide show that NaBH4 reduction of the substrate/product imine complex involves a dramatic decrease in the flexibility of the enzyme, with only very minor changes in the overall secondary and tertiary structure.

3-Methylglutaconic aciduria associated with Pearson syndrome and respiratory chain defects.

3-Methylglutaconic aciduria was detected in four patients with Pearson syndrome, a multitissue disorder with hematologic abnormalities, lactic acidosis resulting from defective oxidative phosphorylation, and deletions in the mitochondrial genome. 3-Methylglutaconic acid may be an additional useful marker for Pearson syndrome and may be a more specific marker than other organic acids identified in this disorder.

An extracellular enzyme from Fusarium solani f. sp. phaseoli which catalyses hydration of the isoflavonoid phytoalexin, phaseollidin.

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) are the prenylated isoflavonoids kievitone and phaseollidin. Two enzyme activities, kievitone hydratase and phaseollidin hydratase, occur in culture filtrates of the bean pathogen, Fusarium solani f. sp. phaseoli, and catalyse similar hydration reactions on the dimethylallyl moieties of the phytoalexins. The enzymes nearly co-purified during hydroxyapatite chromatography followed by preparative native gel electrophoresis. Eluates from successive slices taken from the native gel were assayed for both activities. Although they were not completely separated in the native gel, the activity profiles indicated that the two activities were distinct. The Km of phaseollidin hydratase for phaseollidin was approximately 7 microM.