Novel muteins of human tumor necrosis factor α

For chemical synthesis of a gene coding for human necrosis factor α (TNF-α), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 hp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into vector (]KK223-3) for high expression of active TNF-α in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-α were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-α. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-β. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-α. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.     

In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.     

The effect of tumour necrosis factor-alpha (TNF-alpha) muteins on human neutrophils in vitro

Tumour necrosis factor-alpha (TNF-alpha) has been implicated as an important inflammatory mediator. In vitro, TNF-alpha is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-alpha are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-alpha molecule, four TNF-alpha derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3-7 selected hydrophilic or hydrophobic amino acids in the original TNF-alpha genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-alpha is discussed.     

Conserved low-affinity nickel-binding amino acids are essential for the function of the nickel permease NixA of Helicobacter pylori

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.     

Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.

Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.     

Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli

Replication fidelity is not constant among strains within a species or at all genetic loci within a genome. Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains. We have been studying replication fidelity in Escherichia coli, both in laboratory attenuated strains and in food-borne pathogens. To understand the altered patterns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopolymeric runs of five, seven and nine [G:C] pairs, as well as non-repetitive DNA. Replication of the plasmid in mutS strains resulted in a 20-fold increase in mutant progeny plasmids. The mutations were almost all (>90%) frameshift mutations, while base substitution mutations were rare. Most mutations were insertions or deletions of one or two [G:C] pairs in the longest homopolymeric runs. Larger deletions (5 to >70bp), also targeted to the repetitive sequence, were likewise common. Mutations increased exponentially with the length of the homopolymeric run. These patterns of mutation, including unexpectedly high levels in repair proficient strains, led to an examination of the E. coli K-12 genome for homopolymeric DNA. This sequence motif was found to be rare, particularly in genes and open reading frames. Amino acid homotrimers were found to avoid usage of homopolymeric codons, even when they are preferred among synonymous codons in E. coli. There appears to be active selection against tandem direct nucleotide repeats in the E. coli genome, correlated with the inability of the organism to accurately replicate such sequence.     

DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.     

Expression and Characterization of Fibroblast Growth Factor 8 from Mexican Axolotl, Ambystoma mexicanum

Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface. In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein. The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay. The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4-1.5 fold. To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed. Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites. Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E. coli. The rFGF8 muteins were purified and their binding activities were analyzed. Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity. These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.     

Escherichia coli DNA polymerase IV mutator activity: genetic requirements and mutational specificity

The dinB gene of Escherichia coli is known to be involved in the untargeted mutagenesis of lambda phage. Recently, we have demonstrated that this damage-inducible and SOS-controlled gene encodes a novel DNA polymerase, DNA Pol IV, which is able to dramatically increase the untargeted mutagenesis of F' plasmid. At the amino acid level, DNA Pol IV shares sequence homologies with E. coli UmuC (DNA Pol V), Rev1p, and Rad30p (DNA polymerase eta) of Saccharomyces cerevisiae and human Rad30A (XPV) proteins, all of which are involved in translesion DNA synthesis. To better characterize the Pol IV-dependent untargeted mutagenesis, i.e., the DNA Pol IV mutator activity, we analyzed the genetic requirements of this activity and determined the forward mutation spectrum generated by this protein within the cII gene of lambda phage. The results indicated that the DNA Pol IV mutator activity is independent of polA, polB, recA, umuDC, uvrA, and mutS functions. The analysis of more than 300 independent mutations obtained in the wild-type or mutS background revealed that the mutator activity clearly promotes single-nucleotide substitutions as well as one-base deletions in the ratio of about 1:2. The base changes were strikingly biased for substitutions toward G:C base pairs, and about 70% of them occurred in 5'-GX-3' sequences, where X represents the base (T, A, or C) that is mutated to G. These results are discussed with respect to the recently described biochemical characteristics of DNA Pol IV.     

Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.     

Effectiveness of substituting E. coli DNA-polymerase for DNA-polymerase A in oligonucleotide-directed mutagenesis

The possibility to use the E. coli intact DNA polymerase I in the oligonucleotide-directed site-specific mutagenesis of DNA has been studied. Optimal conditions of the extension activity of this enzyme were found. We have shown that the substitution of the Klenow fragment of the E. coli DNA polymerase by the intact DNA polymerase I did not decrease the efficiency and fidelity of the oligonucleotide-directed mutagenesis.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.     

Biological activities from extracts of endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia

A total of 39 endophytic fungi have been isolated from Viguiera arenaria and Tithonia diversifolia, both collected in S?o Paulo State, Brazil. The isolates were identified based on their ribosomal DNA sequences. The ethyl acetate (EtOAc) extracts of all endophytic fungi were evaluated for their antimicrobial, antiparasitic and antitumoral activity. Antimicrobial screening was conducted using an agar diffusion assay against three pathogenic microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. Antiparasitic activity was determined by enzymatic inhibition of gGAPDH of Trypanosoma cruzi and adenine phosphorybosiltransferase (APRT) of Leishmania tarentolae. Antitumoral activity was tested against human T leukemia cells by the Mosmann colorimetric method. All extracts showed activity in at least one assay: 79.5% of the extracts were cytotoxic against leukemia cells, 5.1% of the extracts were active against S. aureus, 25.6% against E. coli and 64.1% against Candida albicans. Only one extract showed promising results in the inhibition of parasitic enzymes gGAPDH (95.0%) and three were found to inhibit APRT activity. The cytotoxic extract produced by the strain VA1 (Glomerella cingulata) was fractionated and yielded nectriapyrone and tyrosol. Nectriapyrone showed relevant cytotoxic activity against both human T leukemia and melanoma tumor cell lines.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.     

The proton pore of the F0F1-ATPase of Escherichia coli: Ser-206 is not required for proton translocation

A series of experiments was carried out to investigate the role of some polar amino acids in the a-subunit of the ATP synthase of Escherichia coli. Site-directed mutagenesis resulted in the amino acid substitutions Ser-199----Ala, Ser-202----Ala, Ser-206----Ala, Arg-61----Gln or Asp-44----Asn. None of these amino acid substitutions affected the ability of the cells to carry out oxidative phosphorylation. It was concluded therefore that the effect of the substitution of leucine for Ser-206 reported previously (Cain, B.D. and Simoni, R.D. (1986) J. Biol. Chem. 261, 10043-10050) was due to the presence of the leucine rather than the absence of serine. Even though cells carrying the Asp-44----Asn substitution were able to carry out oxidative phosphorylation, membranes from such cells remained proton-impermeable after removal of the F1-ATPase. It appears likely that the proton pore of the F0 of the ATP synthase of E. coli consists of four amino acids, namely Arg-219, Glu-210 and His-245 of the a-subunit and Asp-61 of the c-subunit.     

Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice

BACKGROUND: Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflammatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. METHODS: Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. RESULTS: We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. CONCLUSIONS: The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy.     

Expression and Characterization of Fibroblast Growth Factor 8 from Mexican Axolotl, Ambystoma mexicanum

Fibroblast growth factor (FGF) has been known to regulate the proliferation and differentiation of a variety of cell types via interaction with a specific FGF receptor on the cell surface. In the present study, Fgf8 cDNA of Mexican axolotl, Ambystoma mexicanum, was expressed in Escherichia coli as an MBP-FGF8 fusion protein. The cell proliferation activity of the recombinant FGF8 (rFGF8) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MTT) assay. The addition of rFGF8 to the culture medium enhanced proliferation of BALB/c 3T3 and BHK21 cells about 1.4--1.5 fold. To analyze the binding activity of rFGF8 to the cell surface, cell surface enzyme linked immunosorbent assay was developed. Comparison of the structure of basic FGF with the computer-simulated structure of FGF8 suggested that Tyr-58, Glu-132, Tyr-139, and Leu-179 might be the potential receptor binding sites. Amino acid substitution muteins of FGF8 were constructed by PCR-derived directed mutagenesis and the muteins were overexpressed in E. coli. The rFGF8 muteins were purified and their binding activities were analyzed. Substitution of Tyr-58 or Glu-132 or Leu-179 of the FGF8 with alanine reduced the binding affinity, while substitution of Tyr-139 with alanine did not alter the binding affinity. These results imply that Tyr-58, Glu-132, and Leu-179 of FGF8 might be involved in its binding to the cell surface.     

Characterization of two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln).

Two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln) were expressed in pyruvate dehydrogenase complex-deletion mutant Escherichia coli JRG1342. The expressed mutant E3s were purified to near homogeneity using DEAE-Sephacel and hydroxyapatite columns. The initial velocity measurements in the absence of products for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines in double-reciprocal plots, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its Km for dihydrolipoamide was dramatically increased by 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on double-reciprocal plots. Its specific activity decreased to 28% of that of wild-type enzyme. Its Km for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in human E3 reaction, is critical to human E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.     

Sequence of cloned enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:N-acetylglucosamine phosphotransferase system of Escherichia coli

In Escherichia coli, N-acetylglucosamine (nag) metabolism is joined to glycolysis via three specific enzymes that are the products of the nag operon. The three genes of the operon, nagA, nagB, and nagE, were found to be carried by a colicin plasmid, pLC5-21, from a genomic library of E. coli [Clarke, L., & Carbon, J. (1976) Cell (Cambridge, Mass.) 9,91-99]. The nagE gene that codes for enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was sequenced. The nagE sequence is preceded by a catabolite gene activator protein binding site and ends in a putative rho-independent termination site. The amino acid sequence determined from this DNA sequence shows 44% homology to enzymes IIglucose and IIIglucose of the PTS. Enzyme IIN-acetylglucosamine, which has 648 amino acids and a molecular weight of 68,356, contains a histidine at residue 569 which is homologous to the active site of IIIglc. Sequence homologies with enzymes IIglucose, II beta-glucoside, and IIsucrose indicate that residues His-190, His-213, and His-295 of enzyme IInag are also conserved and that His-190 is probably the second active site histidine. Other sequence homologies among these enzymes II suggest that they contain several sequence transpositions. Preliminary models of the enzymes II are proposed.