Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.     

Reconstitution and characterization of aminopyrrolnitrin oxygenase, a Rieske N-oxygenase that catalyzes unusual arylamine oxidation

Rieske oxygenases catalyze a wide variety of important oxidation reactions. Here we report the characterization of a novel Rieske N-oxygenase, aminopyrrolnitrin oxygenase (PrnD) that catalyzes the unusual oxidation of an arylamine to an arylnitro group. PrnD from Pseudomonas fluorescens Pf5 was functionally expressed in Escherichia coli, and the activity of the purified PrnD was reconstituted, which required in vitro assembly of the Rieske iron-sulfur cluster into the protein and the presence of NADPH, FMN, and an E. coli flavin reductase SsuE. Biochemical and bioinformatics studies indicated that the reconstituted PrnD contains a Rieske iron-sulfur cluster and a mononuclear iron center that are formed by residues Cys(69), Cys(88), His(71), His(91), Asp(323), His(186), and His(191), respectively. The enzyme showed a limited range of substrate specificity and catalyzed the conversion of aminopyrrolnitrin into pyrrolnitrin with K(m) = 191 microM and k(cat) = 6.8 min(-1). Isotope labeling experiments with (18)O(2) and H(2)(18)O suggested that the oxygen atoms in the pyrrolnitrin product are derived exclusively from molecular oxygen. In addition, it was found that the oxygenation of the arylamine substrates catalyzed by PrnD occurs at the enzyme active site and does not involve free radical chain reactions. By analogy to known examples of arylamine oxidation, a catalytic mechanism for the bioconversion of amino pyrrolnitrin into pyrrolnitrin was proposed. Our results should facilitate further mechanistic and crystallographic studies of this arylamine oxygenase and may provide a new enzymatic route for the synthesis of aromatic nitro compounds from their corresponding aromatic amines.     

Purification and properties of L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the formation of beta-alanine in Escherichia coli.

L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the production of beta-alanine, has been purified to apparent homogeneity from Escherichia coli. The properties of the enzyme are: (a) pH optimum of 6.8 to 7.5, (b) temperature optimum of 55 degrees C, (c) Km for L-aspartate of 0.16 mM, and (d) molecular weight of 58,000. The activity of the enzyme is inhibited by reagents (hydroxylamine, phenylhydrazine, and sodium borohydride) that react with carbonyl groups, but no pyridoxal phosphate is present. The compound containing the carbonyl group has been identified as covalently bound pyruvate. Approximately 1 mol of pyruvate was found/mol of enzyme. That the enzyme has a biosynthetic function rather than a catabolic role is indicated by the observations that a mutant (designated as E. coli 99-2) which requires either beta-alanine or pantothenic acid for growth contains only trace amounts of enzyme activity, whereas it is present in substantial amounts in the parent strain (E. coli W) and in a spontaneous revertant of the mutant.     

Molecular cloning, characterization, and downregulation of an acyltransferase that catalyzes the malonylation of flavonoid and naphthol glucosides in tobacco cells

Tobacco cells (Nicotiana tabacum L. Bright Yellow T-13) exposed to harmful naphthols accumulate them as glucosylated and further modified compounds [Taguchi et al. (2003a) Plant Sci. 164, 231-240]. In this study, we identified the accumulated compounds to be 6'-O-malonylated glucosides of naphthols. Cells treated with various phenolic compounds accumulated the flavonoids mainly as malonylglucosides. To clarify the function of this malonylation in tobacco, we isolated the cDNA encoding a malonyltransferase (NtMaT1) from a cDNA library derived from tobacco cells. The heterologous expression of the gene in Escherichia coli revealed that the recombinant enzyme had malonyltransferase activity against several phenolic glucosides such as flavonoid 7-O-glucosides, flavonoid 3-O-glucosides and naphthol glucosides. The substrate preference of the enzyme was similar to that of the tobacco cell extract. Malonylation activity in the transgenic cells markedly decreased with the suppression of the expression of NtMaT1 mRNA in tobacco BY-2 cells by RNA interference. The compounds administered to the transgenic cells were accumulated in the cells as glucosides or other modified compounds in place of malonylglucosides. These results show that NtMaT1 is the main catalyst of malonylation on glucosides of xenobiotic flavonoids and naphthols in tobacco plants.     

Molecular cloning,expression and tissue distribution of hamster diacetyl reductase. Identity with l-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced l-xylulose as well as diacetyl, and slowly oxidized xylitol. The K_m values for l-xylulose and xylitol were similar to those reported for l-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with l-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

Purification, identification, and cloning of lysoplasmalogenase, the enzyme that catalyzes hydrolysis of the vinyl ether bond of lysoplasmalogen

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ～50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ～20 μm). The predominant band on a gel at ～19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.     

Purification and characterization of NAD-dependent morphine 6-dehydrogenase from hamster liver cytosol, a new member of the aldo-keto reductase superfamily

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, was purified 815-fold to a homogeneous protein from the soluble fraction of hamster liver with a yield of 15%. The enzyme was a monomeric protein with a molecular weight of 38 kDa and an isoelectric point of 5.6. Although both NAD and NADP served as cofactors, the enzyme activity with NADP was less than 5% that found with NAD at pH 7.4. With NAD, the enzyme gave the maximal activity at pH 9.3, and the K(m) and V(max) values toward morphine were 1.0 mM and 0.43 unit/mg protein, respectively. Among morphine congeners, normorphine exhibited higher activity than morphine, but codeine and ethylmorphine were poor substrates, and dihydromorphine and dihydrocodeine showed no detectable activity. The enzyme also exhibited significant activity for a variety of cyclic and alicyclic alcohols. In addition to xenobiotics, the enzyme catalyzed the dehydrogenation of 17beta-hydroxysteroids with much higher affinities than morphine. In the reverse reaction, the enzyme exhibited high activity for o-quinones, but morphinone, naloxone, and aromatic aldehydes and ketones were reduced at slow rates. Sulfhydryl reagents and ketamine strongly inhibited the enzyme, whereas pyrazole, barbital, and indomethacin had little effect on enzyme activity. 17beta-Hydroxysteroids inhibited the enzyme in a competitive manner against morphine. A total of 302 amino acid residues, which comprised approximately 94% of whole protein, were identified by sequencing of the peptides obtained by proteolytic digestion. This amino acid sequence of the enzyme showed significant homology to members of the aldo-keto reductase (AKR) superfamily and shared 63-64% identity with members of the AKR1C subfamily. These findings indicate that the enzyme is a new member of the AKR superfamily that is involved in steroid metabolism as 17beta-hydroxysteroid dehydrogenase as well as xenobiotic metabolism.     

Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism

Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.     

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1.

Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2+, oxidized toluene to p-cresol as the first detectable product. Experiments with p-deutero-toluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 18O2, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.     

Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

Molecular cloning and expression of chondroitin 4-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.