Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli ADP-glucose synthetase as deduced from the nucleotide sequence of the glg C gene

The nucleotide sequence of the glg C gene of Escherichia coli K12, coding for ADP-glucose synthetase, has been determined. The structural gene consists of 1293 base pairs, which specify a protein of 431 amino acids. The amino acid sequence deduced from the DNA sequence is consistent with the known NH2-terminal amino acid sequence and the amino acid composition of ADP-glucose synthetase. The translation start of the structural gene of glycogen synthase, glg A, starts immediately after termination of the glg C gene.     

Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli B ADPglucose:1,4-alpha-D-glucan 4-alpha-glucosyltransferase.

The Escherichia coli B glycogen synthase has been purified to apparent homogeneity with the use of a 4-aminobutyl-Sepharose column. Two fractions of the enzyme were obtained: glycogen synthase I with a specific activity of 380 mumol mg-1 and devoid of branching enzyme activity and glycogen synthase II having a specific activity of 505 mumol mg-1 and containing branching enzyme activity which was 0.1% of the activity observed for the glycogen synthase. Only one protein band was found in disc gel electrophoresis for each glycogen synthase fraction and they were coincident with glycogen synthase activity. One major protein band and one very faint protein band which hardly moved into the gel were observed in sodium dodecyl sulfate gel electrophoresis of the glycogen synthase fractions. The subunit molecular weight of the major protein band in sodium dodecyl sulfate gel electrophoresis of both glycogen synthase fractions was determined to be 49 000 +/- 2 000. The molecular weights of the native enzymes were determined by sucrose density gradient ultracentrifugation. Glycogen synthase I had a molecular weight of 93 000 while glycogen synthase II had a molecular weight of 200 000. On standing at 4 degrees C or at -85 degrees C both enzymes transform into species having molecular weights of 98 000, 135 000, and 185 000. Thus active forms of the E. coli B glycogen synthase can exist as dimers, trimers, and tetramers of the subunit. The enzyme was shown to catalyze transfer of glucose from ADPglucose to maltose and to higher oligosaccharides of the maltodextrin series but not to glucose. 1,5-Gluconolactone was shown to be a potent inhibitor of the glycogen synthase reaction. The glycogen synthase reaction was shown to be reversible. Formation of labeled ADPglucose occurred from either [14C]ADP or [14C]glycogen. The ratio of ADP to ADPglucose at equilibrium at 37 degrees C was determined and was found to vary threefold in the pH range of 5.27-6.82. From these data the ratio of ADP2- to ADPglucose at equilibrium was determined to be 45.8 +/- 4.5. Assuming that deltaF degrees of the hydrolysis of the alpha-1,4-glucosidic linkage is -4.0 kcal the deltaF degrees of hydrolysis of the glucosidic linkage in ADPglucose is -6.3 kcal.     

Biosynthesis of bacterial glycogen: primary structure of Salmonella typhimurium ADPglucose synthetase as deduced from the nucleotide sequence of the glgC gene.

The nucleotide sequence of a 1.4-kilobase-pair fragment containing the Salmonella typhimurium LT2 glgC gene coding for ADPglucose synthetase was determined. The glgC structural gene contains 1,293 base pairs, having a coding capacity of 431 amino acids. The amino acid sequence deduced from the nucleotide sequence shows that the molecular weight of ADPglucose synthetase is 45,580. Previous results of the total amino acid composition analysis and amino acid sequencing (M. Lehmann and J. Preiss, J. Bacteriol. 143:120-127, 1980) of the first 27 amino acids from the N terminus agree with that deduced from nucleotide sequencing data. Comparison of the Escherichia coli K-12 and S. typhimurium LT2 ADPglucose synthetase shows that there is 80% homology in their nucleotide sequence and 90% homology in their deduced amino acid sequence. Moreover, the amino acid residues of the putative allosteric sites for the physiological activator fructose bisphosphate (amino acid residue 39) and inhibitor AMP (amino acid residue 114) are identical between the two enzymes. There is also extensive homology in the putative ADPglucose binding site. In both E. coli K-12 and S. typhimurium LT2, the first base of the translational start ATG of glgA overlaps with the third base TAA stop codon of the glgC gene.     

Primary structure of cold-adapted alkaline phosphatase from a Vibrio sp. as deduced from the nucleotide gene sequence

Alkaline phosphatases (AP) are widely distributed in nature, and generally have a dimeric structure. However, there are indications that either monomeric or multimeric bacterial forms may exist. This paper describes the gene sequence of a psychrophilic marine Vibrio AP, previously shown to be particularly heat labile. The kinetic properties were also indicative of cold adaptation. The amino acid sequence of the Vibrio G15-21 AP reveals that the residues involved in the catalytic mechanism, including those ligating the metal ions, have precedence in other characterized APs. Compared with Escherichia coli AP, the two zinc binding sites are identical, whereas the metal binding site, normally occupied by magnesium, is not. Asp-153 and Lys-328 of E. coli AP are His-153 and Trp-328 in Vibrio AP. Two additional stretches of amino acids not present in E. coli AP are found inserted close to the active site of the Vibrio AP. The smaller insert could be accommodated within a dimeric structure, assuming a tertiary structure similar to E. coli AP. In contrast the longer insert would most likely protrude into the interface area, thus preventing dimer formation. This is the first primary structure of a putative monomeric AP, with indications as to the basis for a monomeric existence. Proximity of the large insert loop to the active site may indicate a surrogate role for the second monomer, and may also shape the catalytic as well as stability characteristics of this enzyme.     

Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli

The nitrile hydratase (NHase) of Rhodococcus species N-774, which is composed of two subunits, alpha and beta, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the alpha and beta subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the alpha subunit includes that for the beta subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the alpha- and beta-subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

Biosynthesis of bacterial glycogen. Kinetic studies of a glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) from a glycogen-deficient mutant of Escherichia coli B.

An Escherichia coli B mutant, SG14, accumulates glycogen at 28% the rate observed for the parent E. coli B strain. The glycogen accumulated in the mutant is similar to the glycogen isolated from the parent strain with respect to alpha- and beta-amylosis, chain length determination, and I2-complex absorption spectra. The SG14 mutant contains normal glycogen synthase and branching enzyme activity but has an ADP-glucose pyrophosphorylase with altered kinetic and allosteric properties. The mutant enzyme has been partially purified and requires a 12-fold higher concentration of fructose-P2 or a 26 fold higher concentration of pyridoxal-P than the parent type enzyme for 50% of maximal allosteric activation. TPNH, an effective activator of the E. coli B enzyme, does not activate the SG14 ADP-glucose pyrophosphorylase. Other studies show that for the SG14 enzyme the concentrations of ATP and Mg2+ in the synthesis direction and the concentrations of ADP-glucose and PPi in the pyrophosphorolysis direction required to give 50% of maximal activity are 3- to 6-fold higher than those observed for the parent E. coli B ADP-glucose pyrophosphorylase. The Km for alpha-glucose-1-P at saturating to half-saturating concentrations of the activator, fructose-P2, are about the same for both enzymes. However, in the presence of no activator, the concentration of glucose-1-P required for half-maximal activity is about 1.8-fold higher for the SG14 enzyme. Thus SG14 ADP-glucose pyrophosphorylase has lower affinity for its substrates than does the parent enzyme. Previously the SG14 enzyme had been shown to be less sensitive to inhibition by 5'-AMP than the E. coli B enzyme. This ensensitivity to inhibition renders the SG14 enzyme less responsive to energy charge than the E. coli B ADP-glucose pyrophosphorylase. On the basis of the above results and taking into account the reported concentrations of fructose-P2, of pyridoxal-P, and of the adenine nucleotide pool and its energy charge in E. coli strains, it is concluded that furctose-P2 is the important physiological allosteric activator of E. coli ADP-glucose pyrophosphorylase. Furthermore, the 1.7-fold increased rate of accumulation of glycogen observed when E. coli B or SG14 shifts from exponential phase to stationary phase of growth in nitrogen-limiting media can be accounted for by the 2.4-fold increase of the levels of the glycogen biosynthetic enzymes, glycogen synthase, and ADP-glucose pyrophosphorylase. Thus both allosteric regulation of the ADP-glucose pyrophosphorylase as well as the genetic regulation of the biosynthesis of the glycogen biosynthetic enzymes are involved in the regulation of glycogen accumulation in E. coli B.     

Primary structure of human poly(ADP-ribose) synthetase as deduced from cDNA sequence

Human poly(ADP-ribose) synthetase consists of three proteolytically separable domains, the first for binding of DNA, the second for automodification, and the third for binding of the substrate, NAD (Ushiro, H., Yokoyama, Y., and Shizuta, Y. (1987) J. Biol. Chem. 262, 2352-2357). We have isolated and sequenced cDNA clones for the enzyme using synthesized oligodeoxyribonucleotide probes based on the partial amino acid sequence of the protein. The open reading frame determined encodes a protein of 1,013 amino acid residues with a molecular weight of 113,203. The deduced amino acid sequence is consistent with the partial amino acid sequences of tryptic or alpha-chymotryptic peptides and the total amino acid composition of the purified enzyme. The native enzyme is relatively hydrophilic as judged from the hydrophilicity profile of the total amino acid sequence. The net charge of the NAD binding domain is neutral but the DNA binding domain and the automodification domain are considerably rich in lysine residue and quite basic. The DNA binding domain involves a homologous repeat in the sequence and exhibits a sequence homology with localized regions of transforming proteins such as c-fos and v-fos. Furthermore, this domain contains a unique sequence element which resembles the essential peptide sequences for nuclear location of SV40 and polyoma virus large T antigens. These facts suggest the possibility that the physiological function of poly(ADP-ribose) synthetase lies in its ability to bind to DNA and to control transformation of living eukaryotic cells like the cases of those oncogene products.     

Protein B1 of ribonucleotide reductase. Direct analytical data and comparisons with data indirectly deduced from the nucleotide sequence of the Escherichia coli nrdA gene

The total composition, the N-terminal amino acid sequence, and the amino acid sequences of four internal regions have been determined for the ribonucleotide reductase large subunit, protein B1, prepared from a recombinant lambda-lysogenic Escherichia coli K strain, which overproduces the enzyme 30-50-fold. The data have been compared with those previously reported for B1 prepared from a thymine-starved E. coli B strain and with the indirectly derived primary structure of B1 recently reported from the nucleotide sequence of the E. coli K nrdA gene. Two major differences to these results were found. First, the B1 polypeptides started with initiator Met-1 (45%), Asn-2 (30%) or Gln-3 (15%), demonstrating a different type of N-terminal heterogeneity than that found earlier. Secondly, the total amino acid composition as derived from hydrolyzed protein B1 differed substantially from the amino acid composition derived from the nucleotide data. This has the consequence that Cys, Arg, Thr and possibly Val and Ser appear more frequently whereas Asx, Glx, Tyr and possibly Gly appear less frequently in the nucleotide-derived data as compared to direct protein hydrolysates. We suggest usage of other reading frames in the approximate area of residues 630-700 of the primary structure of the nrdA gene to compensate for these discrepancies and for the relatively high incidence of uncommon codons in the reading frame proposed for this area of the gene. Such changes have implications on the previously assigned putative active-site region of protein B1.     

Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene

We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11. The clones were sequenced and found to encode a protein of 691 amino acid residues with a calculated molecular weight of 76,737 daltons. The amino-terminal sequence (excluding the initial methionine residue) deduced therefrom was in agreement with the protein sequence of the yeast reductase. In addition, the deduced sequence included the partial amino acid sequence determined with the papain-solubilized reductase. The total amino acid sequence of the yeast reductase showed 33-34% similarity with those of the rat, rabbit, pig, and trout reductases. In spite of low similarity in the total amino acid sequences, the possible functional domains related to binding of FAD, FMN, and NADPH were well conserved among all five species compared.     

Primary structure of Bacillus subtilis enolase gene deduced from c-DNA sequence

The nucleotide sequence for enolase gene of Bacillus subtilis was determined from recombinant clone pRE. The sequence was composed of 1570 bp which included the 1374 bp of the complete coding region, the 86 bp of the 5' noncoding region and the 110 bp of the 3' noncoding region containing a polyadenylation signal. In addition, the poly(A) tail was also found. A potential ribosome binding site was located 20 nucleotides upstream of the initiation codon in the 5' noncoding region. The aminoacid sequence deduced from the nucleotide sequence was 558 aminoacids in length. The size of the mRNA was 1.5 kb by the northern transfer technique.     

Branched-chain amino acid aminotransferase of Escherichia coli: nucleotide sequence of the ilvE gene and the deduced amino acid sequence

The ilvE gene of the Escherichia coli K-12 ilvGEDA operon, which encodes branched-chain amino acid aminotransferase [EC 2.6.1.42], was cloned. The nucleotide sequence of 1.5 kilobase pairs containing the gene was determined. The coding region of the ilvE gene contained 927 nucleotide residues and could encode 309 amino acid residues. The predicted molecular weight, amino acid composition and the sequence of the N-terminal 15 residues agreed with the enzyme data reported previously (Lee-Peng, F.-C., et al. (1979) J. Bacteriol. 139, 339-345). From the deduced amino acid sequence, the secondary structure was predicted.     

Complete nucleotide sequence of the Escherichia coli gdhA gene

The DNA sequence of the gdhA gene of Escherichia coli K12, which encodes the 447 amino acid polypeptide subunit of NADP-specific glutamate dehydrogenase, is presented. The deduced protein sequence is strongly homologous to the corresponding enzyme of the eukaryotic fungus Neurospora crassa. The upstream DNA sequence includes several overlapping promoter consensus sequences. The downstream DNA sequence contains inverted repeats, predicted as forming long stable stem-loop structures in RNA, homologous to those found in several enterobacterial intergenic regions.