Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.     

Mechanistic roles of Thr134, Tyr160, and Lys 164 in the reaction catalyzed by dTDP-glucose 4,6-dehydratase

Escherichia coli dTDP-glucose 4,6-dehydratase and UDP-galactose 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family. A highly conserved triad consisting of Ser/Thr, Tyr, and Lys is present in the active sites of these enzymes as well in other SDR proteins. Ser124, Tyr149, and Lys153 in the active site of UDP-galactose 4-epimerase are located in similar positions as the corresponding Thr134, Tyr160, and Lys164, in the active site of dTDP-glucose 4,6-dehydratase. The role of these residues in the first hydride transfer step of the dTDP-glucose 4,6-dehydratase mechanism has been studied by mutagenesis and steady-state kinetic analysis. In all mutants except T134S, the k(cat) values are more than 2 orders of magnitude lower than of wild-type enzyme. The substrate analogue, dTDP-xylose, was used to investigate the effects of the mutations on rate of the first hydride transfer step. The first step becomes significantly rate limiting upon mutation of Tyr160 to Phe and only partly rate limiting in the reaction catalyzed by K164M and T134A dehydratases. The pH dependence of k(cat), the steady-state NADH level, and the fraction of NADH formed with saturating dTDP-xylose show shifts in the pK(a) assigned to Tyr160 to more basic values by mutation of Lys164 and Thr134. The pK(a) of Tyr160, as determined by the pH dependence of NADH formation by dTDP-xylose, is 6.41. Lys164 and Thr134 are believed to play important roles in the stabilization of the anion of Tyr160 in a fashion similar to the roles of the corresponding residues in UDP-galactose 4-epimerase, which facilitate the ionization of Tyr149 in that enzyme [Liu, Y., et al. (1997) Biochemistry 35, 10675--10684]. Tyr160 is presumably the base for the first hydride transfer step, while Thr134 may relay a proton from the sugar to Tyr160.     

The amino acid sequence of the calmodulin obtained from sea anemone (Metridium senile) muscle

The amino acid sequence of the calmodulin obtained from sea anemone muscle was determined. The calmodulin was composed of 148 amino acid residues and its amino terminal was blocked. When compared with bovine brain calmodulin, the number of amino acid residues per molecule was the same and there were 3 replacements at residues 99 (Tyr → Phe), 143 (Gln → Lys) and 147 (Ala → Ser).     

Amino acid sequence of human plasma apolipoprotein C-II from normal and hyperlipoproteinemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-II isolated from normal individuals and a subject with type V hyperlipoproteinemia has been determined. Apo-C-II contains 79 amino acids with the following amino acid composition: Asp4, Asn1, Thr9, Ser9, Glu7, Gln7, Pro4, Gly2, Ala6, Val4, Met2, Ile1, Leu8, Tyr5, Phe2, Lys6, Arg1, Trp1. Cleavage of apo-C-II by cyanogen bromide produced three peptides designated as CB-1 (9 residues), CB-2 (51 residues), and CB-3 (19 residues). Two peptides, CT-1 (50 residues) and CT-2 (29 residues), which overlapped the cyanogen bromide peptides, were obtained by tryptic digestion of citraconylated apo-C-II at the single arginine residue. The amino acid sequence of apo-C-II was obtained by the automated phenyl isothiocyanate degradation of intact apo-C-II and the peptides produced by cleavage of apo-C-II by cyanogen bromide and trypsin. Phenylthiohydantoins were identified by high performance liquid chromatography and chemical ionization-mass spectrometry. The amino acid sequence of apo-C-II from the normal subject was identical with the apo-C-II isolated from the hyperlipoproteinemic subject.     

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis

L-Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 A resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N-terminal residues of subunits related by the P-axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L-xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased K(m) for the substrate (>26-fold) and K(d) for NADPH (95-fold), but only the N107L mutation significantly decreased k(cat) value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism.     

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Spectroscopic characterization of plastocyanin from hornwort

Plastocyanin isolated from an aquatic higher plant, Ceratophyllum demersum L. (hornwort), has been characterized by electronic absorption, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption, CD, and EPR spectra of hornwort plastocyanin indicated a complete similarity of blue copper center to those of terrestrial higher plants and algae. However, the ultraviolet absorption spectrum of hornwort plastocyanin exhibited a lower tyrosine (Tyr) and a higher phenylalanine (Phe) content of the protein comparing with other plastocyanins. The ratio of Phe/Tyr residues was estimated to be 9 by amino acid analysis. The hornwort plastocyanin resembles in amino acid composition terrestrial higher plant plastocyanins rather than alga plastocyanins but is peculiar in the content of Phe and Tyr residues.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides

UDP-galactose 4-epimerase contains the coenzyme NAD+ bound tightly at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+ may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+ bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm to 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 153 to Met results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD+ in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+ in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes the positive electrostatic field and attenuates the reactivity of NAD+. Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD+. The effects of binding the substrate analogue P1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.     

Probing the active site of mitogillin,a fungal ribotoxin

Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G₄₃₂₅ and A₄₃₂₆, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.     

Interaction mechanism between microtubule-associated proteins and microtubules. A proton nuclear magnetic resonance analysis on the binding of synthetic peptide to tubulin

An amino acid sequence essential for microtubule-associated proteins (MAPs) to bind to microtubules is presented [Aizawa et al. (1989) J. Biol. Chem. 264, 5885-5890]. A synthetic peptide of 23 amino acid residues which corresponded to the sequence [tubulin binding peptide (TBP)] was active in binding to tubulin and inducing its assembly. The TBP-tubulin interaction mechanism was analyzed by proton nuclear magnetic resonance spectroscopy as a simplified model for MAP-microtubule interactions. Intraresidue transferred nuclear Overhauser effects (TRNOEs) of TBP in TBP-tubulin mixtures were analyzed, and strong binding of two Val and two Lys residues of TBP to tubulin was detected. Among the sharply peaked signals from tubulin aromatic residues, those due to Tyr ring protons broadened upon mixing with TBP, suggesting the involvement of Tyr residue(s) in the binding with TBP. Irradiation of the tubulin Tyr protons resulted in an intermolecular TRNOE at TBP methyl proton resonances. Evidently, hydrophobic interactions between Val and Tyr residues are important for the binding of TBP to tubulin. Hydrophobic interactions have not been taken into account previously in the widely accepted electrostatic model for the binding of MAPs to microtubules.     

Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus.

The nucleotide sequence of the gene for a highly alkaline, low-molecular-mass pectate lyase (Pel-15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino-acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel-15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site-directed mutagenesis of the Pel-15 gene, we replaced Lys20 in the N-terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C-terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel-15 in the N-terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel-15 in the C-terminal regions. The substituted PelB segment could also express beta-elimination activity in the chimeric molecule, confirming that Pel-15 and PelB share a similar active-site topology.     

一种新的短链脱氢/还原酶家族新成员:NADP(H)-依赖的视黄醇脱氢酶基因的克隆和分析

从牛的肝脏中快速抽提总RNA,根据GenBank已发表NADP(H)-依赖的视黄醇脱氢酶基因(NRDR)的cDNA序列,设计并合成特异引物,利用cDNA末端快速扩增(RACE)方法和反转录-聚合酶链式反应(RT-PCR),得到牛肝内的NRDR cDNA的全长序列。经测序证实,牛肝NRDR的全长cDNA序列为1266bp,其开放读码框架在24～806bp,编码260个氨基酸(GenBank登录号：AF487454)。根据NRDR基因推导出的氨基酸序列与人、鼠、兔有高度同源性,并含有SDR超家族成员的两个高度保守的模序,在其C-端含有过氧化物酶体的靶向序列为SHL。结果表明,牛的NRDR应属于过氧化物酶体内SDR超家族成员并在维甲酸合成的限速步骤起作用的酶,也为维甲酸合成的传统通路提供一个补充。