Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 ± 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 ± 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K_m values were 62 ± 6 and 5.8 ± 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K_m values for glyoxylate and NADH were 5700 ± 600 and 2.9 ± 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.     

Differential expression of two hemA mRNAs encoding glutamyl-tRNA reductase proteins in greening cucumber seedlings.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.     

Isolation and expression of a Bacillus cereus gene encoding benzil reductase.

Benzil was reduced stereospecifically to (S)-benzoin by Bacillus cereus strain Tim-r01. To isolate the gene responsible for asymmetric reduction, we constructed a library consisting of Escherichia coli clones that harbored plasmids expressing Bacillus cereus genes. The library was screened using the halo formation assay, and one clone showed benzil reduction to (S)-benzoin. Thus, this clone seemed to carry a plasmid encoding a Bacillus cereus benzil reductase. The deduced amino acid sequence had marked homologies to the Bacillus subtilis yueD protein (41% identity), the yeast open reading frame YIR036C protein (31%), and the mammalian sepiapterin reductases (28% to 30%), suggesting that benzil reductase is a novel short-chain de-hydrogenases/ reductase.     

Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase.

The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame of 987 bp with a 225-bp 3'-untranslated region. The 328-amino acid protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial species.     

马克斯克鲁维酵母羰基还原酶基因的克隆与表达

【目的】研究羰基还原酶基因的克隆、表达及其在不对称生物催化中的应用。【方法】对羰基还原酶氨基酸序列进行BLAST推导出核苷酸序列,设计引物,以马克斯克鲁维酵母(Kluyveromyce marxianus)CGMCC 2.1977全基因组为模板,通过PCR扩增目的片段,与载体pET-28a连接,转化大肠杆菌获得重组菌BL21(DE3)-(pET28a-cMCR)和Rosetta(DE3)-(pET28a-cMCR)。【结果】扩增的序列与已报道的mer序列有100%同源性,全长1 038 bp,共编码345个氨基酸。目的蛋白在Rosetta(DE3)-(pET28a-cMCR)得到了高效表达,大小为42 kD。该酶最适反应温度为40°C,最适反应pH是8,热稳定性与pH稳定性较差。Ca2+对酶活具有明显的激活作用,且浓度为0.5 mmol/L时效果最好。重组菌可还原4-氯乙酰乙酸乙酯(COBE)为(S)-4-氯-3-羟基丁酸乙酯[(S)-CHBE],光学纯度为100%,转化率为81.0%。重组菌在制备度洛西汀关键中间体(S)-氮,氮-二甲基-3-羟基-(2-噻吩)-l-丙胺[(S)-DHTP]中也得到初步应用。【结论】从菌株马克斯克鲁维酵母(Kluyveromyce marxianus)CGMCC 2.1977中克隆获得了羰基还原酶基因,在大肠杆菌中成功表达,并可应用于不对称还原。     

不同波长光源对新蚜虫疠霉产孢能力的影响及霉菌蓝光受体基因的克隆和表达分析

光在调控真菌的多种生理过程中发挥着重要作用。为探究光照对新蚜虫疠霉Pandora neoaphidis产孢的影响,本文研究了不同波长光源(蓝光、绿光、白光、红光、黄光)和无光黑暗条件对新蚜虫疠霉分生孢子弹射能力的影响,通过cDNA末端的快速扩增(RACE)对新蚜虫疠霉的蓝光受体蛋白基因pnwc-1进行克隆并对其进行生物信息学分析,利用qRT-PCR对蓝光光源不同照射时长下pnwc-1的表达量进行了定量分析。结果表明,蓝色光源(波长460–465nm)照射后的新蚜虫疠霉菌丝产生的分生孢子数量显著高于其他波长光源,排序为:蓝光>绿光>白光>红光>黄光>无光。另外,分析克隆获得的全长为2 423bp的pnwc-1基因发现,其编码的蛋白具有蓝光受体蛋白典型的保守结构域,同源比对结果显示新蚜虫疠霉与接合菌门真菌归为一类但相对独立。qRT-PCR的定量分析结果表明随着照射时长增加,蓝光处理能显著提高pnwc-1的表达量,而且pnwc-1的相对表达量与累积产孢量存在正相关(R2=0.9798)。本研究为后续蓝光及其受体基因功能的深入研究提供了实验基础,并促进以新蚜虫疠霉为代表的虫霉目真菌在害虫生物防治中的应用。     

Enzymology of the reduction of hydroxypyruvate and glyoxylate in a mutant of barley lacking peroxisomal hydroxypyruvate reductase

The use of LaPr 88/29 mutant of barley (Hordeum vulgare), which lacks NADH-preferring hydroxypyruvate reductase (HPR-1), allowed for an unequivocal demonstration of at least two related NADPH-preferring reductases in this species: HPR-2, reactive with both hydroxypyruvate and glyoxylate, and the glyoxylate specific reductase (GR-1). Antibodies against spinach HPR-1 recognized barley HPR-1 and partially reacted with barley HPR-2, but not GR-1, as demonstrated by Western immunoblotting and immunoprecipitation of proteins from crude leaf extracts. The mutant was deficient in HPR-1 protein. In partially purified preparations, the activities of HPR-1, HPR-2, and GR-1 could be differentiated by substrate kinetics and/or inhibition studies. Apparent K_m values of HPR-2 for hydroxypyruvate and glyoxylate were 0.7 and 1.1 millimolar, respectively, while the K_m of GR-1 for glyoxylate was 0.07 millimolar. The K_m values of HPR-1, measured in wild type, for hydroxypyruvate and glyoxylate were 0.12 and 20 millimolar, respectively. Tartronate and P-hydroxypyruvate acted as selective uncompetitive inhibitors of HPR-2 (K_i values of 0.3 and 0.4 millimolar, respectively), while acetohydroxamate selectively inhibited GR-1 activity. Nonspecific contributions of HPR-1 reactions in assays of HPR-2 and GR-1 activities were quantified by a direct comparison of rates in preparations from wild-type and LaPr 88/29 plants. The data are evaluated with respect to previous reports on plant HPR and GR activities and with respect to optimal assay procedures for individual HPR-1, HPR-2, and GR-1 rates in leaf preparations.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1.

The gene encoding the serine cycle hydroxypyruvate reductase of Methylobacterium extorquens AM1 was isolated by using a synthetic oligonucleotide with a sequence based on a known N-terminal amino acid sequence. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced a serine cycle hydroxypyruvate reductase null mutant. This mutant had lost its ability to grow on C-1 compounds but retained the ability to grow on C-2 compounds, showing that the hydroxypyruvate reductase operating in the serine cycle is not involved in the conversion of acetyl coenzyme A to glycine as previously proposed. A second hydroxypyruvate-reducing enzyme with a low level of activity was found in M. extorquens AM1; this enzyme was able to interconvert glyoxylate and glycollate. The gene encoding hydroxypyruvate reductase was shown to be located about 3 kb upstream of two other serine cycles genes encoding phosphoenolpyruvate carboxylase and malyl coenzyme A lyase.     

Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus.

Functional disruption of the gene encoding nitrate reductase (niaD) in Aspergillus parasiticus was conducted by two strategies, one-step gene replacement and the integrative disruption. Plasmid pPN-1, in which an internal DNA fragment of the niaD gene was replaced by a functional gene encoding orotidine monophosphate decarboxylase (pyrG), was constructed. Plasmid pPN-1 was introduced in linear form into A. parasiticus CS10 (ver-1 wh-1 pyrG) by transformation. Approximately 25% of the uridine prototrophic transformants (pyrG+) were chlorate resistant (Chlr), demonstrating their inability to utilize nitrate as a sole nitrogen source. The genetic block in nitrate utilization was confirmed to occur in the niaD gene by the absence of growth of the A. parasiticus CS10 transformants on medium containing nitrate as the sole nitrogen source and the ability to grow on several alternative nitrogen sources. Southern hybridization analysis of Chlr transformants demonstrated that the resident niaD locus was replaced by the nonfunctional allele in pPN-1. To generate an integrative disruption vector (pSKPYRG), an internal fragment of the niaD gene was subcloned into a plasmid containing the pyrG gene as a selectable marker. Circular pSKPYRG was transformed into A. parasiticus CS10. Chlr pyrG+ transformants were screened for nitrate utilization and by Southern hybridization analysis. Integrative disruption of the genomic niaD gene occurred in less than 2% of the transformants. Three gene replacement disruption transformants and two integrative disruption transformants were tested for mitotic stability after growth under nonselective conditions. All five transformants were found to stably retain the Chlr phenotype after growth on nonselective medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.