Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene

The nucleotide sequence of the mtlA gene, which codes for the mannitol-specific Enzyme II of the Escherichia coli phosphotransferase system, is presented. From the gene sequence, the primary translation product is predicted to consist of 637 amino acids (Mr = 67,893). This result is compared to the amino acid composition and molecular weight of the purified mannitol Enzyme II protein. The hydrophobic and hydrophilic properties of the enzyme were evaluated along its amino acid sequence using a computer program (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). The computer analysis predicts that the NH2-terminal half of the enzyme resides within the membrane, whereas the COOH-terminal half of the enzyme has the properties of a soluble protein. The possible functions of such a protein structure are discussed. RNA mapping has identified the promoter and mRNA start point for the mtl operon.     

Amino acid sequence studies on sheep liver fructose-bisphosphatase. II. The complete sequence

The cyanogen bromide fragments of S-carboxymethylated fructose-bisphosphatase were purified. The amino acid sequences of the small fragments were determined by the dansyl-Edman method. The large fragments were subjected to proteolytic digestion to give smaller peptides more amenable for purification and sequencing by similar methods. Enzyme digests of the S-carboxymethylated enzyme gave overlap peptides containing the methionine residues. In conjunction with the amino acid sequence of the 60-residue N-terminal fragment previously determined on the S-peptide released by limited proteolysis with subtilisin the complete sequence of 336 residues was deduced. The sequence has been compared with the 335 residue sequence of pig kidney fructose-bisphosphatase and some areas of sequence for rabbit liver enzyme. The strong homology previously noted for the S-peptide sequence is maintained for the complete enzyme with only 34 changes in 336 residues when comparing the pig and sheep enzymes.     

The ENZYME data bank.

The ENZYME data bank is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) and it contains the following data for each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided: EC number Recommended name Alternative names (if any) Catalytic activity Cofactors (if any) Pointers to the SWISS-PROT protein sequence entrie(s) that correspond to the enzyme (if any) Pointers to human disease(s) associated with a deficiency of the enzyme (if any).     

Subunit association of enzyme I of the Salmonella typhimurium phosphoenolpyruvate: glycose phosphotransferase system. Temperature dependence and thermodynamic properties

The bacterial phosphoenolpyruvate:glycose phosphotransferase system plays an essential role in diverse physiological phenomena. To perform these functions, the system is stringently regulated, although the underlying molecular regulatory mechanisms have not been established. A potential target for this type of regulation is the first protein in the phosphotransfer sequence, Enzyme I, which catalyzes the following reaction: P-enolpyruvate + Enzyme I Mg2+ in equilibrium phospho-I + pyruvate. We reported previously that Enzyme I from Salmonella typhimurium consists of identical subunits which associate in a temperature-dependent manner; the mode of association was found to be either monomer-dimer or isodesmic. The association reaction has now been investigated by analytical gel chromatography at 8, 11, and 23 degrees C. At each temperature, the mode of association was strictly monomer-dimer. The apparent association equilibrium constant, K'a, increased dramatically with temperature, with an enthalpy of 54.8 +/- 6.3 kcal/mol. At 23 degrees C, K'a decreased slightly when the enzyme solution contained either Mg2+ or phosphoenolpyruvate. However, when both ligands were present, i.e. under conditions where Enzyme I is phosphorylated, K'a decreased significantly (25-fold at 11 degrees C and 50-fold at 23 degrees C). These results are in accord with a model for the action of Enzyme I which involves a cycle of association and dissociation. This model has potentially important implications for regulating Enzyme I and the bacterial phosphoenolpyruvate:glycose phosphotransferase system.     

Using GO-PseAA predictor to predict enzyme sub-class

Enzyme function is much less conserved than anticipated, i.e., the requirement for sequence similarity that implies similarity in enzymatic function is much higher than the requirement that implies similarity in protein structure. This is because the function of an enzyme is an extremely complicated problem that may involve very subtle structural details as well as many other physical chemistry factors. Accordingly, if simply based on the sequence similarity approach, it would hardly get a decent success rate in predicting enzyme sub-class even for a dataset consisting of samples with 50% sequence identity. To cope with such a situation, the GO-PseAA predictor was adopted to identify the sub-class for each of the six main enzyme families. It has been observed that, even for the much more stringent datasets in which none of the enzymes has 25% sequence identity to any others, the overall success rates are 73-95%, suggesting that the GO-PseAA predictor can catch the core features of the statistical samples concerned and may become a useful high throughput tool in proteomics and bioinformatics.     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.     

Predicting enzyme family classes by hybridizing gene product composition and pseudo-amino acid composition

A new method has been developed to predict the enzymatic attribute of proteins by hybridizing the gene product composition and pseudo amino acid composition. As a demonstration, a working dataset was generated with a cutoff of 60% sequence identity to avoid redundancy and bias in statistical prediction. The dataset thus constructed contains 39989 protein sequences, of which 27469 are non-enzymes and 12520 enzymes that were further classified into 6 enzyme family classes according to their 6 main EC (Enzyme Commission) numbers (2314 are oxidoreductases, 3653 transferases, 3246 hydrolases, 1307 lyases, 676 isomerases, and 1324 ligases). The overall success rate by the jackknife test for the identification between enzyme and non-enzyme was 94%, and that for the identification among the 6 enzyme family classes was 98%. It is anticipated that, with the rapid increase of protein sequences entering into databanks, the current method will become a useful automated tool in identifying the enzymatic attribute of a newly found protein sequence.     

Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate- (PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140 000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70 000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [32P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group.     

Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum

A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized.     

A survey of orphan enzyme activities

Background  

Using computational database searches, we have demonstrated previously that no gene sequences could be found for at least 36% of enzyme activities that have been assigned an Enzyme Commission number. Here we present a follow-up literature-based survey involving a statistically significant sample of such "orphan" activities. The survey was intended to determine whether sequences for these enzyme activities are truly unknown, or whether these sequences are absent from the public sequence databases but can be found in the literature.     

How well is enzyme function conserved as a function of pairwise sequence identity?

Enzyme function conservation has been used to derive the threshold of sequence identity necessary to transfer function from a protein of known function to an unknown protein. Using pairwise sequence comparison, several studies suggested that when the sequence identity is above 40%, enzyme function is well conserved. In contrast, Rost argued that because of database bias, the results from such simple pairwise comparisons might be misleading. Thus, by grouping enzyme sequences into families based on sequence similarity and selecting representative sequences for comparison, he showed that enzyme function starts to diverge quickly when the sequence identity is below 70%. Here, we employ a strategy similar to Rost's to reduce the database bias; however, we classify enzyme families based not only on sequence similarity, but also on functional similarity, i.e. sequences in each family must have the same four digits or the same first three digits of the enzyme commission (EC) number. Furthermore, instead of selecting representative sequences for comparison, we calculate the function conservation of each enzyme family and then average the degree of enzyme function conservation across all enzyme families. Our analysis suggests that for functional transferability, 40% sequence identity can still be used as a confident threshold to transfer the first three digits of an EC number; however, to transfer all four digits of an EC number, above 60% sequence identity is needed to have at least 90% accuracy. Moreover, when PSI-BLAST is used, the magnitude of the E-value is found to be weakly correlated with the extent of enzyme function conservation in the third iteration of PSI-BLAST. As a result, functional annotation based on the E-values from PSI-BLAST should be used with caution. We also show that by employing an enzyme family-specific sequence identity threshold above which 100% functional conservation is required, functional inference of unknown sequences can be accurately accomplished. However, this comes at a cost: those true positive sequences below this threshold cannot be uniquely identified.     

Regulation of Glutamine Synthetase X. Effect of Growth Conditions on the Susceptibility of Escherichia coli Glutamine Synthetase to Feedback Inhibition

The kinetic properties of Escherichia coli glutamine synthetase are markedly influenced by the manner in which the organism is grown. Enzyme obtained from stationary-phase cells grown on glycerol and glutamate is strongely inhibited by each of the eight feedback effectors known to influence this enzyme; however, the enzyme from log-phase cells grown on glucose and growth-limiting concentrations of NH(4)Cl is stimulated by some of these effectors. Of the growth variables examined, nitrogen source and time of harvest were the most important; carbon source and aeration seemed to have no effect. Two purified enzyme preparations have been obtained from cells grown under two different conditions, designated enzymes I and II for convenience. Enzyme I is stimulated by adenosine 5'-monophosphate, histidine, and tryptophan in the transfer assay, whereas enzyme II is strongly inhibited by all effectors tested. Enzyme I has a higher specific activity in the forward assay in the presence of Mg(++) or Co(++), whereas enzyme II is more active in the presence of Mn(++).     

EFICAz: a comprehensive approach for accurate genome-scale enzyme function inference

EFICAz (Enzyme Function Inference by Combined Approach) is an automatic engine for large-scale enzyme function inference that combines predictions from four different methods developed and optimized to achieve high prediction accuracy: (i) recognition of functionally discriminating residues (FDRs) in enzyme families obtained by a Conservation-controlled HMM Iterative procedure for Enzyme Family classification (CHIEFc), (ii) pairwise sequence comparison using a family specific Sequence Identity Threshold, (iii) recognition of FDRs in Multiple Pfam enzyme families, and (iv) recognition of multiple Prosite patterns of high specificity. For FDR (i.e. conserved positions in an enzyme family that discriminate between true and false members of the family) identification, we have developed an Evolutionary Footprinting method that uses evolutionary information from homofunctional and heterofunctional multiple sequence alignments associated with an enzyme family. The FDRs show a significant correlation with annotated active site residues. In a jackknife test, EFICAz shows high accuracy (92%) and sensitivity (82%) for predicting four EC digits in testing sequences that are <40% identical to any member of the corresponding training set. Applied to Escherichia coli genome, EFICAz assigns more detailed enzymatic function than KEGG, and generates numerous novel predictions.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Evidence for the evolutionary relatedness of the proteins of the bacterial phosphoenolpyruvate:sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) found in enteric bacteria is a complex enzyme system consisting of a non-sugar-specific phosphotransfer protein called Enzyme I, two small non-sugar-specific phosphocarrier substrates of Enzyme I, designated HPr and FPr, and at least 11 sugar-specific Enzymes II or Enzyme II-III pairs which are phosphorylated at the expense of phospho-HPr or phospho-FPr. In this communication, evidence is presented which suggests that these proteins share a common evolutionary origin and that a fructose-specific phosphotransferase may have been the primordial ancestor of them all. The evidence results from an evaluation of 1) PTS protein sequence data; 2) structural analysis of operons encoding proteins of the PTS; 3) genetic regulatory mechanisms controlling expression of these operons; 4) enzymatic characteristics of the PTS systems; 5) immunological cross reactivities of these proteins; 6) comparative studies of phosphotransferase systems from evolutionarily divergent bacteria; 7) the nature of the phosphorylated protein intermediates; 8) molecular weight comparisons among the different Enzymes II and Enzyme II-III pairs; and 9) interaction studies involving different PTS protein constituents. The evidence leads to a unifying theory concerning the evolutionary origin of the system, explains many structural, functional, and regulatory properties of the phosphotransferase system, and leads to specific predictions which should guide future research concerned with genetic, biochemical, and physiological aspects of the system.     

Expression and characterization of a novel class of glutathione S-transferase from Anopheles dirus

A new Anopheles dirus glutathione S-transferase (GST) has been obtained and named adGST4-1. Both genomic DNA and cDNA for heterologous expression were acquired. The genomic sequence was 3188bp and consisted of the GST gene as well as flanking sequence. The flanking sequence was analyzed for possible regulatory elements that would control gene expression. In Drosophila several of these elements have been shown to be involved in development and cell differentiation. The deduced amino acid sequence has low identity compared with the four alternatively spliced enzymes, adGST1-1 to 1-4, from another An. dirus GST gene adgst1AS1. The percent identities are 30--40% and 11--12% comparing adGST4-1 to insect GSTs from Delta and Sigma classes, respectively. Enzyme characterization of adGST4-1 shows it to be distinct from the other An. dirus GSTs because of low enzyme activity for customary GST substrates including 1-chloro-2, 4-dinitrobenzene (CDNB). However, this enzyme has a greater affinity of interaction with pyrethroids compared to the other An. dirus GSTs.     

Comparative study of methodologies for obtaining β-glucosidase immobilized on dextran-modified silica

In this study, two procedures for the immobilization of β-glucosidase on silica are compared. The first approach comprises a preliminary stabilization of β-glucosidase by coupling with dextran dialdehyde and subsequent immobilization of the obtained β-glucosidase dextran dialdehyde with aminopropylsilica. In the second approach, β-glucosidase is immobilized on silica modified with a dextran-dialdehyde coating. Enzyme immobilized via coupling with dextran dialdehyde and subsequent attachment with aminopropylsilica show a remarkably enhanced thermostability. Enzyme immobilized by the alternative approach demonstrated an inferior thermoresistance. The difference in behavior of the immobilized enzyme obtained via these two methods can be explained considering the number of links between the enzyme and carrier. Enzyme immobilized on dextran dialdehydecoated silica is fixed via a limited number of links. On the other hand, with soluble β-glucosidase-dextran conjugates, the enzyme configuration is already stabilized via a high number of links with the dextran backbone. It is clear from this study that the sequence of reactions in immobilizing enzymes on silica support via a dextran-dialdehyde linker has a significant effect on the final properties.     

Glucitol-specific enzymes of the phosphotransferase system in Escherichia coli. Nucleotide sequence of the gut operon

The complete nucleotide sequence of the glucitol (gut) operon in Escherichia coli has been determined. The glucitol-specific Enzyme II and Enzyme III of the phosphoenolpyruvate:sugar phosphotransferase system as well as glucitol-6-phosphate dehydrogenase which are encoded by the gutA, gutB, and gutD genes of the gut operon, respectively, are predicted to consist of 506 (Mr = 54,018), 123 (Mr = 13,306), and 259 (Mr = 27,866) amino acyl residues, respectively. The hydropathic profile of the Enzyme IIgut revealed 7 or 8 long hydrophobic segments which may traverse the cell membrane as alpha-helices as well as 2 or 4 short strongly hydrophobic stretches which may traverse the membrane as beta-structure. The number of amino acyl residues in the sum of the molecular weights of the glucitol Enzyme II-III pair are nearly the same as those of the mannitol Enzyme II. The ratio of hydrophobic to hydrophilic amino acyl residues and the numbers of the hydrophobic segments are also nearly the same for both transport systems. However, no significant homology was found in the nucleotide or amino acyl sequences of the two systems. Glucitol-6-phosphate dehydrogenase was found to exhibit sequence homology with ribitol dehydrogenase. A repetitive extragenic palindromic sequence was found in the 3'-flanking region of the gutD gene, suggesting the presence of a gene downstream from the gutD gene.     

Amino acid sequence of an active site peptide of avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Hydroxymethylglutaryl-CoA synthase is irreversibly inhibited by the active site-directed inhibitor 3-chloropropionyl-CoA. Enzyme modification has been postulated to involve alkylation of an active site cysteinyl sulfhydryl group. DEAE-Sephadex chromatography of tryptic digests prepared from enzyme inactivated using chloro[14C]propionyl-CoA suggested that bound radioactivity is localized on one peptide. Specificity of the modification was further demonstrated by reverse-phase high pressure liquid chromatography, which was used to isolate the radioactively labeled peptide in a chemically homogeneous form. Automated gas-phase Edman degradation techniques have been employed to confirm the assignment of cysteine as the inhibitor's target residue and to elucidate the sequence of amino acids which flank the 14C-carboxyethylated cysteine: Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-(Thr)- Asn-Ala-S-[14C]carboxyethylcysteine-Tyr-Gly-Gln-Thr-(Ala). These data represent the first assignment of active site structure for hydroxymethyl-glutaryl-CoA synthase.