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PubMed1995

Substrate specificity of nine NAD(+)-dependent alcohol dehydrogenases in Aspergillus nidulans.

In Aspergillus nidulans three alcohol dehydrogenases (ADHs) have been described. ADHI is induced by ethanol and is the physiological enzyme of ethanol utilization, ADHII has not been attributed a function but is repressed by ethanol. The ALCR regulatory protein acts positively to induce ADHI, and negatively in its control of ADHII. ADHIII is specifically induced by anaerobic stress. We have characterized the substrate specificity of these three enzymes by looking at their staining profile on polyacrylamide gels with a range of alcohols. In addition to these enzymes we have observed six other NAD(+)-dependent ADHs, two of which, propan-2-ol dehydrogenase and pentan-2-ol dehydrogenase, share similar control with ADHII. The inducibility of these enzymes with some alcohols has also been investigated. The profile of ADHs with NADP+ as an electron acceptor is also reported.

Alcohol Dehydrogenase↗

PubMed2001

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Amino Acid Substitution↗

PubMed1994

Substrate specificity of the NS3 serine proteinase of hepatitis C virus as determined by mutagenesis at the NS3/NS4A junction.

Hepatitis C virus (HCV) encodes a polyprotein that is processed to produce the structural and nonstructural proteins of the virus. Nonstructural protein 3 (NS3) is a serine proteinase that cleaves the polyprotein to release the NS4A, NS4B, NS5A, and NS5B proteins. To characterize the substrate specificity of NS3, we synthesized by in vitro translation the polyprotein NS2*-NS3-NS4*P that includes 70% of the NS2 protein, the complete NS3 protein, and 25% of the NS4 protein region attached to substance P, an epitope tag. We demonstrated that NS3 cleaves at the NS3/NS4A junction to release the NS4*P protein. Subsequently, we used this reaction to evaluate the importance of conserved amino acids that flank the NS3/NS4A junction. We replaced amino acids in the P6, P1, and P1' positions of the scissile bond of this junction using site-directed mutagenesis. When the P6 aspartic acid was changed to asparagine, lysine, or serine, NS3-mediated cleavage occurred. When threonine in the P1 position was replaced with other polar amino acids or with amino acids having aliphatic side chains, cleavage occurred, although it was not detected when arginine or tyrosine was present. Replacement of serine in the P1' position with other polar amino acids, with amino acids having aliphatic side chains, or with arginine resulted in NS3-mediated cleavage. Thus, since fewer amino acids in the P1 position supported cleavage than in the P6 or P1' positions, the P1 position of the scissile bond may play a more important role in defining the substrate specificity of the HCV NS3 proteinase.

Amino Acid Sequence↗

PubMed1993

Substrate-specific enhancement of the oxidative half-reaction of monoamine oxidase.

Monoamine oxidases A and B have identical flavin sites but different, although overlapping, amine substrate specificity. Reoxidation of ternary complexes containing substrate is much faster than of free enzyme, and the enhancement is greater in the A form than the B form. The oxidative half-reaction was studied with a variety of substrates to elucidate the specificity of the effect and to probe the different influences of substrate on the flavin reoxidation in the two forms of the enzyme. The second-order rate constant for the reoxidation was highest with monoamine oxidase A when kynuramine was the ligand (508 x 10(3) M-1 s-1) compared to 4 x 10(3) M-1 s-1 in its absence. MPTP (166 x 10(3) M-1 s-1) also enhanced reoxidation well, but indole substrates stimulated only poorly (e.g., tryptamine, 29 x 10(3) M-1 s-1; serotonin, 50 x 10(3) M-1 s-1). For the A form, the reduction of the flavin was rate-limiting in all cases. For the B form, reoxidation was rate-limiting for beta-phenylethylamine and contributed to the determination of the overall rate with several substrates. The ratio of the enhanced rate of oxidation to the rate of reduction correlated with the redox state of the enzyme in turnover experiments. All the observations are consistent with alternate paths of reoxidation, via either free enzyme or a reduced enzyme-substrate complex. The flux through each path is determined by the relative dissociation constants and rate constants.

Animals↗

PubMed1996

Substrate specificity of acetylxylan esterase from Schizophyllum commune: mode of action on acetylated carbohydrates.

Substrate specificity of a purified acetylxylan esterase from Schizophyllum commune was investigated on a variety of methyl per-O-acetyl glycopyranosides, methyl di-O-acetyl-beta-D-xylopyranosides and acetylated polysaccharides. The enzyme preferentially deacetylated the 3-position of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside. Removal of the 3-acetyl group from the xylopyranoside was accompanied by a slower deacetylation at positions 2 and 4. A similarly slower, accompanying deacetylation occurred primarily at position 2 with the glucopyranoside. Such specificity corresponds well to the expected function of the esterase in acetylxylan degradation. Of the three possible diacetates of methyl beta-D-xylopyranoside, the 3,4-diacetate was found to be the most rapidly deacetylated. Unexpectedly, products of its deacetylation were a mixture of 2- and 4-monoacetate. The formation of the methyl 2-O-acetyl-beta-D-xylopyranoside involved an enzyme-mediated acetyl group transfer because the rate of the enzyme-catalyzed reaction exceeded the rate of spontaneous migration of acetyl groups. This is the likely mechanism for acetyl removal from position 2 in the native substrate. The enzyme exhibited the highest regioselectivity with methyl 2,3,4,6-tetra-O-acetyl-beta-D-mannopyranoside. An 80% conversion of this substrate to methyl 4,6-di-O-acetyl-beta-D-mannopyranoside, a new mannose derivative, was achieved. In contrast to the majority of lipases and esterases exploited for regioselective deacetylation, the S. commune acetylxylan esterase did not attack the C-6 acetyl linkages in methyl hexopyranosides when other acetyl groups were available.

Acetates↗

PubMed2003

Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism.

Treatment with the dioxygenase inhibitor prohexadione-Ca leads to major changes in the flavonoid metabolism of apple (Malus domestica) and pear (Pyrus communis) leaves. Accumulation of unusual 3-deoxyflavonoids is observed, which have been linked to an enhanced resistance toward fire blight. The committed step in this pathway is the reduction of flavanones. Crude extracts from leaves are able to perform this reaction. There was previous evidence that DFR enzymes of certain plants possess additional flavanone 4-reductase (FNR) activity. Such an FNR activity of DFR enzymes is proved here by heterologous expression of the enzymes. The heterologously expressed DFR/FNR enzymes of Malus and Pyrus possess distinct differences in substrate specificities despite only minor differences of the amino acid sequences. Kinetic studies showed that dihydroflavonols generally are the preferred substrates. However, with the observed substrate specificities the occurrence of 3-deoxyflavonoids in vivo after application of prohexadione-Ca can be explained.

Alcohol Oxidoreductases↗

PubMed2003

Biochemical properties and substrate specificities of alkaline and histidine acid phytases.

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium-phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.

6-Phytase↗

PubMed1995

Vesicular L-glutamate transporter in microvesicles from bovine pineal glands. Driving force, mechanism of chloride anion activation, and substrate specificity.

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl- concentration, being negligible in the absence of Cl- and maximum at 2-5 mM and decreasing gradually at 20-100 mM. The membrane potential (inside positive) was maximum at 0-10 mM Cl- and then decreased gradually depending on the Cl- concentration, whereas a pH gradient was practically absent without Cl- and increased gradually up to 100 mM Cl-. Ammonium acetate or nigericin plus K+, a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K+ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP- and Cl(-)-dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H(+)-ATPase being affected. An anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, similarly inhibited the glutamate uptake in a Cl- protectable manner. Furthermore, ATP- and glutamate-dependent acidification of MVs was observed when 4 mM Cl- was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl- regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.

ATP-Binding Cassette Transporters↗

PubMed1996

Arylamine N-acetyltransferases. Expression in Escherichia coli, purification, and substrate specificities of recombinant hamster monomorphic and polymorphic isozymes.

Two isozymes of arylamine N-acetyltransferases (NATs) catalyze the biotransformation of arylamines to arylamides, and the bioactivation of carcinogenic arylhydroxylamines and arylhydroxamic acids to reactive electrophiles capable of forming a variety of DNA and protein adducts. As part of a project directed toward delineation of the molecular factors responsible for the pronounced differences in the substrate specificity of the isozymes, we have recently reported the expression in Escherichia coli and purification of hamster NAT1 (NAT1 8) as a fusion protein to an antibody-reactive amino terminus FLAG peptide capable of being removed by digestion with enterokinase. Unfortunately, the conditions necessary for the removal of the peptide by enterokinase resulted in incomplete protease digestion and substantial loss of NAT1 activity. Consequently, we have constructed the plasmid pPH8 in which an 11 amino acid thrombin proteolysis site has been inserted between the FLAG peptide and the amino terminus of NAT1. In addition, a plasmid that expresses hamster NAT2 (NAT2 15) was constructed by exchanging the gene sequence for NAT1 with the cloned sequence for NAT2. Both NAT fusion proteins were expressed in JM105 cells. Analysis of catalytically active cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that approximately 30-fold more soluble NAT2 was expressed than NAT1 in the bacterial cytosol. The fusion proteins were purified to homogeneity by immunoaffinity chromatography, followed by gel filtration to remove high molecular weight contaminants. The FLAG peptide was subsequently removed by treatment with human thrombin, followed by an additional pass over the immunoaffinity column. Unlike the results obtained from proteolysis by enterokinase, the activities of the rNAT1 and rNAT2 were shown to be unaffected by treatment with thrombin. Moreover, the substrate specificities for the recombinant NATs closely matched those observed for NAT1 and NAT2 isolated from hamster liver.

Animals↗

PubMed1981

The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities.

The MgATP-dependent phosphorylase phosphatase was found to have a broad substrate specificity. Its activity against all phosphoproteins tested was dependent upon preincubation with the activating factor FA and MgATP. The enzyme dephosphorylated and inactivated phosphorylase kinase and inhibitor 1, and dephosphorylated and activated glycogen synthase and acetyl-CoA carboxylase. Glycogen synthase was dephosphorylated at similar rates whether it had been phosphorylated by cyclic-AMP-dependent protein kinase, phosphorylase kinase or glycogen synthase kinase 3. The enzyme also catalysed the dephosphorylation of ATP citrate lyase, initiation factor eIF-2, and troponin I. The properties of the MgATP-dependent protein phosphatase from either dog liver or rabbit skeletal muscle showed a remarkable similarity to highly purified preparations of protein phosphatase 1 from rabbit skeletal muscle. The relative activities of the two enzymes against all phosphoproteins tested was very similar. Both enzymes dephosphorylated the beta-subunit of phosphorylase kinase 40-fold faster than the alpha-subunit, and both enzymes were inhibited by identical concentrations of the two proteins termed inhibitor 1 and inhibitor 2, which inhibit protein phosphatase 1 specifically. These results demonstrate that the MgATP-dependent protein phosphatase is a type-1 protein phosphatase, and is distinct from type-2 protein phosphatases which dephosphorylate the alpha-subunit of phosphorylase kinase and are unaffected by inhibitor 1 and inhibitor 2. The possibility that the MgATP-dependent protein phosphatase is an inactive form of protein phosphatase 1 and that both proteins share the same catalytic subunit is discussed.

Adenosine Triphosphate↗

PubMed1997

Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles.

A novel homologue of p38 MAP kinase, called SAPK4, has been cloned which shares 61% amino acid identity with p38 and is expressed predominantly in testes, pancreas and small intestine. We also cloned an alternative form of p38beta, termed p38beta2, which lacks the additional 8 amino acid insertion unique to p38beta. p38, p38beta, p38beta2, ERK6/p38gamma/SAPK3, and SAPK4 were characterized with respect to stimulus-dependent activation in transfected cells, substrate specificity, and sensitivity to inhibition by pyridinyl imidazoles. All homologues were stimulated, although to differing extents, by IL-1beta, TNF, sorbitol, and UV. Only SAPK3 and SAPK4 were stimulated significantly by PMA. p38beta showed the weakest activation overall. MBP, ATF-2, and both MAPKAP kinase-2 and kinase-3 were good substrates of p38 and p38beta in vitro. In contrast, only MBP, ATF2, and MAPKAP kinase-3 proved to be significant substrates of SAPK3 and SAPK4, and of these three, MAPKAP kinase-3 was by far the weakest. p38beta had very poor kinase activity for all substrates except MBP. While both p38 and p38beta2 were comparably inhibited by SB 203580 and SB 202190, neither SAPK3 nor SAPK4 were inhibited. p38beta was partially inhibited by both inhibitors. These data suggest that SAPK3 and SAPK4 form a distinct subset of the p38 MAP kinases with different expression pattern, response to stimuli, substrate specificity, and inhibitor sensitivity.

Activating Transcription Factor 2↗

PubMed2006

Activity and substrate specificity of pyrimidine phosphorylases and their role in fluoropyrimidine sensitivity in colon cancer cell lines.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) are often upregulated in solid tumors and catalyze the phosphorolysis of natural (deoxy)nucleosides and a wide variety of fluorinated pyrimidine nucleosides. Because the relative contribution of each of the two enzymes to these reactions is still largely unknown, we investigated the substrate specificity of TP and UP in colon cancer cells for the (fluoro)pyrimidine nucleosides thymidine (TdR), uridine (Urd), 5'-deoxy-5-fluorouridine (5'DFUR), and 5FU. Specific inhibitors of TP (TPI) and UP (BAU) were used to determine the contribution of each enzyme in relation to their cytotoxic effect. The high TP expressing Colo320TP1 cells were most sensitive to 5'DFUR and 5FU, with IC50 values of 1.4 and 0.2 microM, respectively, while SW948 and SW1398 were insensitive to 5'DFUR (IC50>150 microM for 5'DFUR). TPI and BAU only moderately affected sensitivity of Colo320, SW948, and SW1398, whereas TPI significantly increased IC(50) for 5'DFUR (50-fold) and 5FU (11-fold) in Colo320TP1 and BAU that in C26A (9-fold for 5'DFUR; p<0.01). In the epithelial skin cell line HaCaT both inhibitors were able to decrease sensitivity to 5'DFUR and 5FU separately. HaCaT might be a model for 5'DFUR toxicity. In the colon cancer cells 5'DFUR degradation varied from 0.4 to 50 nmol 5FU/h/10(6)cells, that of TdR from 0.3 to 103 nmol thymine/h/10(6)cells, that of Urd from 0.8 to 79 nmol uracil/h/10(6)cells, while conversion of 5FU to FUrd was from 0.3 to 46 nmol/h/10(6)cells. SW948 and SW1398 were about equally sensitive to 5'DFUR and 5FU, but SW1398 had higher phosphorylase activity (>65-fold) compared to SW948. In SW948 and HaCaT TPI and BAU inhibited TdR and Urd phosphorolysis (>80%), respectively. Both TP and UP contributed to the phosphorolysis of 5'DFUR and 5FU. In the presence of both inhibitors, still phosphorolysis of 5FU (>40%) was detected in the tumor and HaCaT cell lines, and remarkably, that of all four substrates in SW1398 cells. 5'DFUR phosphorolysis was also measured in situ, where Colo320TP1, SW1398, and HaCaT cells produced significant amounts 5FU from 5'DFUR (>10 nmol/24h/10(6)cells). In Colo320TP1 and in HaCaT cells TPI completely prevented 5FU production, but not in SW1398 cells, where BAU decreased this by 67% (p<0.01). High uracil and dUrd levels were detected in the medium. Uracil accumulation was heavily reduced in the presence of TPI for Colo320TP1 and HaCaT cells, whereas 5FU-induced dUrd production by these cell lines increased (p<0.01). In contrast, for SW1398 cells only BAU was able to reduce uracil levels, and dUrd production remained unchanged. In conclusion, overlapping substrate specificity was found for TP and UP in the cell lines, in which both enzymes were responsible for converting TdR and Urd, and 5'DFUR. 5'DFUR and 5FU were converted to their products in both the colon cancer cells and keratinocytes.

Cell Line, Tumor↗

PubMed1992

Broad substrate specificity of snake venom fibrinolytic enzymes: possible role in haemorrhage.

We found previously that two fibrinolytic enzymes (jararafibrases I and II) purified from Bothrops jararaca venom displayed a haemorrhagic activity. To elucidate the mechanisms involved and the role of the enzymatic activity in haemorrhage, the enzymatic properties of the purified enzymes were examined. The substrate specificity of the enzymes was determined using type I collagen, type IV collagen, gelatin, laminin and fibronectin as substrates. The enzymes degraded type IV collagen, gelatin, laminin and fibronectin into smaller fragments, but degraded type I collagen only partially in a non-specific manner. The specific activities of jararafibrase I for type IV collagen and gelatin were 172 +/- 5 units/mg protein and 1315 +/- 177 units/mg protein, respectively. The specific activities of jararafibrase II for type IV collagen and gelatin were 9.2 +/- 0.6 units/mg protein and 143 +/- 15 units/mg protein, respectively. It was evident that the enzymes had rather broad substrate specificities and degraded basement membrane components including type IV collagen. The number of type IV collagen units of bacterial collagenase which gave the minimal haemorrhagic dose was 191.4, while the numbers of type IV collagenase units of jararafibrases I and II which gave the minimal haemorrhagic dose were 1.5 and 0.25, respectively. It is suggested that the broad substrate specificity of the enzymes is essential for inducing haemorrhage with a single enzyme.

Animals↗

PubMed1986

Change of substrate specificity of rat liver microsomal fatty acyl-CoA synthetase activity by Triton X-100.

The effect of Triton X-100 on the activities and apparent molecular size of fatty acyl-CoA synthetase, solubilized and partially purified from rat liver microsomes, was studied. In the presence of Triton X-100, the activity for lignoceroyl-CoA synthesis was decreased, but activity was restored when the detergent was removed. The appearance and disappearance of lignoceroyl-CoA synthesis appeared related to the size of the aggregated form of the enzyme. On the other hand, activity for palmitoyl-CoA synthesis was not significantly affected by the detergent. Because available evidence suggests that both fatty acids are converted to CoA esters by the same enzyme, it seems likely that the substrate specificity of the enzyme is influenced by changes in the aggregation state and that the microenvironment of the enzyme in membranes may determine the substrate specificity of acyl-CoA synthetase.

Animals↗

PubMed1998

Substrate specificity of organic cation/H+ exchange in avian renal brush-border membranes.

The substrate specificity of the avian renal organic cation exchanger was examined in isolated renal brush-border membrane vesicles. Endobiotic and xenobiotic organic cations (OCs) were tested at a concentration of 100 microM for cis-inhibition of 14C-tetraethylammonium (TEA)/H+ exchange and at 1 mM for trans-stimulation of 14C-TEA efflux. The xenobiotic cations amiloride, cimetidine, mepiperphenidol, procainamide, quinidine, quinine, and ranitidine cis-inhibited TEA uptake >/= 80%; isoproterenol and unlabeled TEA inhibited uptake at least 30%. In contrast, the endogenous cations acetylcholine, choline, and guanidine did not inhibit TEA uptake; however, epinephrine, N1-methylnicotinamide, serotonin, and thiamine inhibited uptake as much as 60%. Each endogenous cation, except thiamine, trans-stimulated TEA efflux, and xenobiotic cations, excluding isoproterenol and TEA, trans-inhibited TEA efflux. The data suggest that the avian renal tubule luminal OC exchanger has greater affinity for xenobiotic cations than for endobiotic cations, but greater transport capacity for endobiotics than for xenobiotics.

Animals↗

PubMed2001

Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120.

Eukaryotic-like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen-fixing and heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic-like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.

Amino Acid Sequence↗

PubMed1999

Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.

DNA↗

PubMed2000

Redefined substrate specificity of ST6GalNAc II: a second candidate sialyl-Tn synthase.

The acceptor substrate specificities of ST6GalNAc I and II, which act on the synthesis of O-linked oligosaccharides, were reexamined using ovine submaxillary mucin, [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11). It has been suggested that only ST6GalNAc I can synthesize carbohydrate structures of sialyl-Tn-antigen; i.e., NeuAc alpha2-6GalNAc-O-Thr/Ser [Kurosawa et al., J. Biol. Chem. 269, 19048-19053 (1994)] based on the result that ST6GalNAc I, not ST6GalNAc II, exhibited activity toward asialoagalacto-fetuin. In this study, we present evidence that both ST6GalNAc I and II exhibit activity toward asialo-OSM (ovine submaxillary mucin) and [Ala-Thr(GalNAc)-Ala]n polymer (n = 7-11) which have only the GalNAc-O-Thr/Ser-structures. These results strongly indicate that not only ST6GalNAc I but also II are candidates for sialyl-Tn synthases.

Animals↗