Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase,partial purification of the enzyme from maturing seeds,and steady-state kinetic analysis of raffinose synthesis

Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.     

Purification and characterization of an N-acylaminoacyl-peptide hydrolase from rabbit muscle

An N-acylaminoacyl-peptide hydrolase has been purified to homogeneity (7,000-fold with 20% yield) from rabbit muscle. This overall enrichment and its general properties as a soluble protein suggest that it is of cytosolic origin and not a component of ribosomes or other cellular organelles. The enzyme has an Mr of 230,000-245,000 and a subunit Mr of 76,000-80,000. An extensive survey of the substrate specificity of the pure enzyme reveals that our earlier conclusions (Radhakrishna, G., and Wold, F. (1986) J. Biol. Chem. 261, 9572-9575) that the enzyme is specific for Ac-Met-peptides are wrong. The enzyme catalyzes the rapid removal of Ac-Thr, Ac-Ala, Ac-Met, Ac-Ser, and more slowly Ac-Gly from peptides of different lengths. Other acetylated amino acids (Cys, Tyr, Asp, Val, Phe, Ile, Leu) may be removed at 1% or less of the rate of the above good substrates from some peptide substrates. The nature of the amino acid in the second position of the acetylated peptide generally has only a minor effect on the reaction rate; however, with charged amino acids (Arg, Asp) in the second position the reaction is retarded, and with proline it is virtually abolished. Except for slow rate of hydrolysis of acetylated dipeptides, the hydrolase does not appear to be severely affected by the peptide length in the range studied (from 2 to 11 amino acid residues). The hydrolase also cleaves formylamino acids from formylated peptides. The biological function of the enzyme is not clear.     

Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.     

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.     

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family

Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani

The gene encoding S-adenosylhomocysteine (AdoHcy) hydrolase in Leishmania donovani was subcloned into an expression vector (pPROK-1) and expressed in Escherichia coli. Recombinant L. donovani AdoHcy hydrolase was then purified from cell-free extracts of E. coli using three chromatographic steps (DEAE-cellulose chromatofocusing, Sephacryl S-300 gel filtration, and Q-Sepharose ion exchange). The purified recombinant L. donovani enzyme exists as a tetramer with a molecular weight of approximately 48 kDa for each subunit. Unlike recombinant human AdoHcy hydrolase, the catalytic activity of the recombinant L. donovani enzyme was shown to be dependent on the concentration of NAD+ in the incubation medium. The dissociation constant (Kd) for NAD+ with the L. donovani enzyme was estimated to be 2.1 +/- 0.2 microM. The Km values for the natural substrates of the enzyme, AdoHcy, Ado, and Hcy, were determined to be 21 +/- 3, 8 +/- 2, and 82 +/- 5 microM, respectively. Several nucleosides and carbocyclic nucleosides were tested for their inhibitory effects on this parasitic enzyme, and the results suggested that L. donovani AdoHcy hydrolase has structural requirements for binding inhibitors different than those of the human enzyme. Thus, it may be possible to eventually exploit these differences to design specific inhibitors of this parasitic enzyme as potential antiparasitic agents.     

Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli

Menaquinone is a lipid-soluble molecule that plays an essential role as an electron carrier in the respiratory chain of many bacteria. We have previously shown that its biosynthesis in Escherichia coli involves a new intermediate, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and requires an additional enzyme to convert this intermediate into (1 R,6 R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). Here, we report the identification and characterization of MenH (or YfbB), an enzyme previously proposed to catalyze a late step in menaquinone biosynthesis, as the SHCHC synthase. The synthase catalyzes a proton abstraction reaction that results in 2,5-elimination of pyruvate from SEPHCHC and the formation of SHCHC. It is an efficient enzyme ( k cat/ K M = 2.0 x 10 (7) M (-1) s (-1)) that provides a smaller transition-state stabilization than other enzymes catalyzing proton abstraction from carbon acids. Despite its lack of the proposed thioesterase activity, the SHCHC synthase is homologous to the well-characterized C-C bond hydrolase MhpC. The crystallographic structure of the Vibrio cholerae MenH protein closely resembles that of MhpC and contains a Ser-His-Asp triad typical of serine proteases. Interestingly, this triad is conserved in all MenH proteins and is essential for the SHCHC synthase activity. Mutational analysis found that the catalytic efficiency of the E. coli protein is reduced by 1.4 x 10 (3), 2.1 x 10 (5), and 9.3 x 10 (3) folds when alanine replaces serine, histidine, and aspartate of the triad, respectively. These results show that the SHCHC synthase is closely related to alpha/beta hydrolases but catalyzes a reaction mechanistically distinct from all known hydrolase reactions.     

Purification and characterization of a beta-D-mannosidase from the marine anaspidean Aplysia fasciata

A beta-D-mannosidase was purified to homogeneity from visceral mass extract of Aplysia fasciata a mollusc belonging to the order Anaspidea. The purified enzyme is a homodimer with a subunit mass of 130 kDa. Temperature and pH optima of this enzyme were 45 degrees C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. The K(M) and V(max) values for p-nitrophenyl beta-D-mannopyranoside were determined to be 2.4 mM and 50.3 micromol min(-1)mg(-1), respectively. The catalytic efficiency of this beta-mannosidase (11,519 min(-1)) was significantly higher than those reported for beta-mannosidases from other sources. It was verified that this is an exo-acting glycosyl hydrolase with transglycosidase activity. When the enzyme was incubated in the presence of p-nitrophenyl beta-D-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a beta-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl alpha-D-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl beta-D-mannopyranoside, two regioisomers (85:15, 12% yield) due to the beta-mannosylation of the heteroacceptor in 4 and in 6 positions were formed.     

Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.     

Properties of liver microsomal cholesterol 5,6-oxide hydrolase

Cholestane 3 beta,5 alpha, 6 beta-triol has been identified as the exclusive product formed on hydration of cholesterol 5,6 alpha- and 5,6 beta-oxide catalyzed by cholesterol oxide hydrolase in liver microsomes obtained from five mammalian species. Highest activities were present in microsomes from rats and humans. Both acid- and base-catalyzed hydrolysis of the two epoxides also produce this product, presumably due to preference for pseudo-axial opening of the oxirane ring to form product with a trans-AB ring junction. Although the beta-oxide is more reactive than the alpha-oxide upon acid-catalyzed hydration, the alpha-oxide is a 4.5-fold better substrate than the beta-oxide as indicated by values of Vmax/Km. The kinetic parameters Vmax and Km for the reaction catalyzed by rat liver microsomes are 1.68 +/- 0.15 X 10(-7) M min-1 and 10.6 +/- 1.5 microM for the alpha-oxide and 1.32 +/- 0.11 X 10(-7) M min-1 and 37.2 +/- 5.5 microM for the beta-oxide at 0.35 mg protein/ml, pH 7.4, 6.35% (v/v) CH3CN, and 37 degrees C. Several imino compounds are competitive inhibitors for the enzyme from rat liver. The most effective of these is 5,6 alpha-iminocholestanol (Ki = 0.085 microM) which was known to be a good inhibitor from previous studies. Inhibition by aziridines is consistent with the participation of acid catalysis in the mechanism of action of the enzyme. Cholesterol oxide hydrolase is a distinct enzyme from oxidosqualene cyclase as well as microsomal epoxide hydrolase (EC 3.3.2.3) and the recently reported mouse hepatic microsomal epoxide hydrolase that catalyzes the hydration of trans-stilbene oxide.     

Molecular characterization of a novel trehalose-6-phosphate hydrolase, TreA, from Bacillus licheniformis

An unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9 kDa. The temperature and pH optima for BlTreA were 30 °C and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-d-glucopyranoside (pNPG) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a K(M) value of 5.2mM and a k(cat) value of 30.2s(-1). Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5-10% acetone and 10-20% ethanol, whereas 5-10% SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.14 M guanidine hydrochloride (GdnHCl) and reached the unfolded intermediates, [GdnHCl](0.5,N-I) and [GdnHCl](0.5,I-U), at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8M urea with [urea](0.5,N-U) of 4.98 M, corresponding to a free energy change of 4.29 kcal/mol for the N→U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. Taken together, the characterization data provide a foundation for the future structure-function studies of BlTreA, a typical member of glycoside hydrolase family 13.     

Acetopyruvate hydrolase production by Pseudomonas putida O1--optimization of batch and fed-batch fermentations

The main objective of this work was the optimization of the production of the beta-ketolase, acetopyruvate hydrolase, from Pseudomonas putida O1. Orcinol was used as an inducer for enzyme production. The growth medium was optimized in two steps. In the first step, screening for optimal glucose concentration was performed. In the second step, a central composite design was used to optimize carbon and nitrogen sources in the medium. After this optimization procedure, a medium was obtained which produced seven times more biomass than the initial medium. Acetopyruvate hydrolase enzyme production was optimized by determining the optimal time of feed and amount of orcinol, using statistical methods. In a subsequent step, the maximal orcinol-degradation rate was determined. The results obtained were used to find an optimal feeding profile for enzyme production. By using the optimized fed-batch process, acetopyruvate hydrolase activity was enhanced from 10 units l(-1)to 400 units l(-1), in comparison with previously reported fermentation experiments. Productivity could even be increased by a factor of 75, to a value of 20 units l(-1 )h(-1).     

Aspartate 142 is involved in both hydrolase and dehydrogenase catalytic centers of 10-formyltetrahydrofolate dehydrogenase

The enzyme 10-formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. N(t)-FDH shares some sequence identity with several 10-formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp(142) in the case of N(t)-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in N(t)-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type N(t)-FDH. Six other aspartate residues located near the conserved Asp(142) were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp(142) is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.     

Purification and characterization of S-adenosylhomocysteine hydrolase from mouse mastocytoma P-815 cells. Evidence for cell cycle-specific fluctuation of the enzyme activity

S-Adenosylhomocysteine hydrolase [EC 3.3.1.1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190,000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45,000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The Km value for adenosine was 0.29 microM and the Vmax value 4.5 mumol S-adenosylhomocysteine X min-1 X mg-1 in the synthetic reaction, while the Km value for S-adenosylhomocysteine was 0.77 microM and the Vmax 0.48 mumol adenosine X min-1 X mg-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (KD = 2.61 X 10(-7) M) and one for cAMP (KD = 1.6 X 10(-7) M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G1/S phase to the G2 phase.     

Biosynthesis and hydrolysis of cholesteryl esters by rat skin subcellular fractions. Regulation by prostaglandins

The properties and subcellular distribution of the enzymes involved with the synthesis and hydrolysis of cholesteryl esters were investigated in skin of normal and essential fatty acid-deficient rats. Most of the activity of the cholesterol-esterifying enzyme(s) is associated with the 12000g and 105000g particulate fractions. The dependence of the enzyme reaction on ATP and CoA suggests that the esterification of cholesterol by rat skin is mediated by a fatty acyl-CoA-cholesterol acyltransferase (EC 2.3.1.-). On the other hand, most of the activity of the cholesteryl ester hydrolase (EC 3.1.1.13) is localized in the 105000g supernatant fraction. Although the activity of the cholesterol-esterifying enzyme(s) was elevated in skin preparations from essential fatty acid-deficient rats, the activity of the hydrolase was significantly decreased. These observations may explain in part the elevated concentrations of sterol esters in the skin of these animals. Prostaglandin E(2) at low concentrations exerted marked inhibitory effect on the activity of the cholesterol-esterifying enzyme(s), whereas no effect was observed on the activity of the hydrolase at similar concentrations. However, at high concentrations prostaglandin E(2) exerted moderate stimulatory effect on the activity of the hydrolase. These results suggest a possible physiological role of this substance in regulating the production of sterol esters in this tissue.     

PLP catabolism: identification of the 2-(Acetamidomethylene)succinate hydrolase gene in Mesorhizobium loti MAFF303099

The function of the mlr6787 gene from Mesorhizobium loti MAFF303099 has been identified. This gene encodes 2-(acetamidomethylene)succinate hydrolase, an enzyme involved in the catabolism of pyridoxal 5'-phosphate (vitamin B 6). This enzyme was overexpressed in Escherichia coli, purified to homogeneity, and characterized. 2-(Acetamidomethylene)succinate hydrolase catalyzes the hydrolysis of 2-(acetamidomethylene)succinate to yield succinic semialdehyde, acetic acid, carbon dioxide, and ammonia. The k cat and K M for this reaction were 0.6 s (-1) and 143 microM, respectively. The enzyme was shown to utilize the E isomer of 2-(acetamidomethylene)succinate.     

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.     

Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme

Epoxide hydrolases are enzymes involved in metabolism and defense of plants. Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana. To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed. We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis. We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A. thaliana. A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose. The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis. The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively. The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C. Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively. The activity was 50-fold lower towards cis-stilbene oxide. The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.