D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.     

DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.     

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.     

Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.     

Purification and characterization of DNA ligase III from bovine testes. Homology with DNA ligase II and vaccinia DNA ligase

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3' mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.     

Non-natural aglycones of glycopeptide antibiotics of the vancomycin group. Synthesis and antibacterial activity

A new approach for the modification of the heptapeptide core of glycopeptide antibiotics was proposed based on the replacement of amino acid residues in positions 1 and 3 in teicoplanin aglycone and in position 1 in the eremomycin aglycone. Six novel nonnatural aglycones of the vancomycin type were obtained. Compounds derived from the teicoplanin aglycone exhibited in vitro activity against Gram-positive bacteria, and two of them were also active against the vancomycin-resistant enterococci.     

Major changes in the kinetic mechanism of AMP inhibition and AMP cooperativity attend the mutation of Arg49 in fructose-1,6-bisphosphatase

The significance of subunit interface residues Arg49 and Lys50 in the function of porcine liver fructose-1,6-bisphosphatase was explored by site-directed mutagenesis, initial rate kinetics, and circular dichroism spectroscopy. The Lys50 --> Met mutant had kinetic properties similar to the wild-type enzyme but was more thermostable. Mutants Arg49 --> Leu, Arg49 --> Asp, Arg49 --> Cys were less thermostable than the wild-type enzyme yet exhibited wild-type values for kcat and Km. The Ki for the competitive inhibitor fructose 2,6-bisphosphate increased 3- and 5-fold in Arg49 --> Leu and Arg49 --> Asp, respectively. The Ka for Mg2+ increased 4-8-fold for the Arg49 mutants, with no alteration in the cooperativity of Mg2+ binding. Position 49 mutants had 4-10-fold lower AMP affinity. Most significantly, the mechanism of AMP inhibition with respect to fructose 1,6-bisphosphate changed from noncompetitive (wild-type enzyme) to competitive (Arg49 --> Leu and Arg49 --> Asp mutants) and to uncompetitive (Arg49 --> Cys mutant). In addition, AMP cooperativity was absent in the Arg49 mutants. The R and T-state circular dichroism spectra of the position 49 mutants were identical and superimposable on only the R-state spectrum of the wild-type enzyme. Changes from noncompetitive to competitive inhibition by AMP can be accommodated within the framework of a steady-state Random Bi Bi mechanism. The appearance of uncompetitive inhibition, however, suggests that a more complex mechanism may be necessary to account for the kinetic properties of the enzyme.     

The history of radium in medicine in Manchester

An established senescence-accelerated model mouse strain, SAMP8, shows the deterioration of learning and memory compared with a normal control strain, SAMR1. D-Serine binds to strychnine-insensitive glycine binding sites of the N-methyl-D-aspartate (NMDA) receptor complex, and enhances glutamate binding to the receptor complex. To investigate the relationship of endogenous brain D-serine and the brain dysfunction caused by aging, the level of brain free D-serine and the D-[3H]serine binding to the brain samples were examined using the SAMP8 and SAMR1 mice. The free D-serine level was highest in the cerebral frontal and occipital cortices in both the SAMP8 and SAMR1; no difference in the D-serine level was shown between the two strains. A receptor autoradiographical analysis showed that the D-[3H]serine binding to the brain section was highest in the hippocampus, and the binding in the SAMP8 brains was lower than that of the SAMR1. The D-[3H]serine binding to the crude cerebral membranes indicated that the value of the total binding sites for the SAMP8 was lower than that for the SAMR1, whereas the value of the dissociation constant Kd for the SAMP8 was similar to that of the SAMR1. These results suggest that the number of D-[3H]serine binding sites was decreased in the SAMP8 compared to the SAMR1, but the affinity of D-[3H]serine to the binding sites was not altered. These results support the view that a decrease of NMDA receptor complex is involved in the age-related neural dysfunction of SAMP8 mice.     

Glycopeptide antibiotic resistance genes in glycopeptide-producing organisms

The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce glycopeptide antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all glycopeptide producers tested. These results suggest that glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.     

In vivo evidence for the link between L- and D-serine metabolism in rat cerebral cortex

To obtain an insight into the metabolic pathways of endogenous D-serine in mammalian brains, we have investigated in the infant rat the effects of systemic administration of L-serine, D-serine, and related amino acids, including glycine and threonine, on the amino acid contents in the cerebral cortex. Intraperitoneal injection of L-serine induced a rapid and transient elevation of the levels of L-serine itself in the neocortex, with its peak at 3 h post injection, and a delayed and prolonged increase in D-serine contents from 1.5 h to at least 24 h thereafter. Similarly, a significant augmentation in cerebral D-serine contents was observed 6 h after intraperitoneal administration of glycine, which also elevated the cortical L-serine levels. In contrast, L-threonine injection affected the concentrations of neither D- nor L-serine in the cortex of the pups. D-Serine given systemically, in turn, increased the neocortical contents of L-serine as well as D-serine itself, but failed to alter those of glycine and L-threonine. These in vivo data suggest the possible link between metabolic pathways of D- and L-serine in the cerebral cortex of the rat.     

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.     

Facile liquid chromatographic enantioresolution of native amino acids and peptides using a teicoplanin chiral stationary phase

The glycopeptide antibiotic teicoplanin is shown to be a highly effective stationary phase chiral selector for the resolution of underivatized amino-acid and imino-acid enantiomers. Fifty four of these compounds (including all chiral protein amino acids) as well as a number of dipeptides were resolved. Hydro-organic mobile phases are used and no buffers or added salts are needed in most cases. Hence the purified analytes are easily isolated in pure form, if needed, by evaporating of the solvent. The effect of pH, organic modifier type and amount are discussed. The enantioselective separation mechanism is examined using both molecular modeling and retention data. The strongest stereoselective interaction is for carboxy-terminated D-amino-acids. In case of peptides, it is not necessary for these to be a D-, D-, terminal sequence for strong interactions. In some cases, including Ala-Ala, the L-, D-, terminal sequence showed greater interaction with the teicoplanin chiral stationary phase.     

Two different allelic mutations in a Finnish family with lecithin:cholesterol acyltransferase deficiency

Lecithin:cholesterol acyltransferase (LCAT) deficiency is a genetic disorder associated with low levels of serum HDL cholesterol. The proband of the Finnish LCAT-deficient family had corneal opacities, proteinuria, anemia with stomatocytosis, low serum HDL cholesterol (0.27 mmol/L), and low LCAT activity. Sequence analysis of his LCAT gene revealed compound heterozygosity for two different mutations: a C insertion in exon 1 between nucleotides 932 and 937 and a C-to-T point mutation in exon 6 at position 4976. The C insertion in exon 1 is predicted to result in premature termination and a truncated polypeptide containing only 16 amino acids. The C-to-T point mutation in exon 6 substitutes cysteine for arginine at residue 399. The functional significance of the Arg399-->Cys mutation was examined by expressing the mutated and wild-type LCAT cDNAs in COS cells. COS cells transfected with mutated and wild-type cDNAs showed comparable levels of mature LCAT mRNA. However, LCAT activity in the cell media of COS cells transfected with the mutant LCAT cDNA was significantly lower than that of COS cells transfected with the wild-type cDNA (1.4% versus 12.0% cholesterol esterified, respectively). A polymerase chain reaction-based duplex assay, in which both mutations can be detected simultaneously, was used for preliminary screening of Finnish subjects with serum HDL levels below 0.9 mmol/L; two additional individuals heterozygous for the Arg399-->Cys mutation were identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.     

Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.     

The lantibiotic mersacidin inhibits peptidoglycan synthesis by targeting lipid II

The lantibiotic mersacidin exerts its bactericidal action by inhibition of peptidoglycan biosynthesis. It interferes with the membrane-associated transglycosylation reaction; during this step the ultimate monomeric peptidoglycan precursor, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-GlcNAc (lipid II) is converted into polymeric nascent peptidoglycan. In the present study we demonstrate that the molecular basis of this inhibition is the interaction of mersacidin with lipid II. The adsorption of [14C]mersacidin to growing cells, as well as to isolated membranes capable of in vitro peptidoglycan synthesis, was strictly dependent on the availability of lipid II, and antibiotic inhibitors of lipid II formation strongly interfered with this binding. Direct evidence for the interaction was provided by studies with isolated lipid II. [14C]mersacidin associated tightly with [14C]lipid II micelles; the complex was stable even in the presence of 1% sodium dodecyl sulfate. Furthermore, the addition of isolated lipid II to the culture broth efficiently antagonized the bactericidal activity of mersacidin. In contrast to the glycopeptide antibiotics, complex formation does not involve the C-terminal D-alanyl-D-alanine moiety of the lipid intermediate. Thus, the interaction of mersacidin with lipid II apparently occurs via a binding site which is not targeted by any antibiotic currently in use.     

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-alpha forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-beta, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.     

Structural studies of receptor binding by cholera toxin mutants

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.