The Tertiary Origin of the Allosteric Activation of E. coli Glucosamine-6-Phosphate Deaminase Studied by Sol-Gel Nanoencapsulation of Its T Conformer

The role of tertiary conformational changes associated to ligand binding was explored using the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase from Escherichia coli (EcGNPDA) as an experimental model. This is an enzyme of amino sugar catabolism that deaminates GlcN6P, giving fructose 6-phosphate and ammonia, and is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P). We resorted to the nanoencapsulation of this enzyme in wet silica sol-gels for studying the role of intrasubunit local mobility in its allosteric activation under the suppression of quaternary transition. The gel-trapped enzyme lost its characteristic homotropic cooperativity while keeping its catalytic properties and the allosteric activation by GlcNAc6P. The nanoencapsulation keeps the enzyme in the T quaternary conformation, making possible the study of its allosteric activation under a condition that is not possible to attain in a soluble phase. The involved local transition was slowed down by nanoencapsulation, thus easing the fluorometric analysis of its relaxation kinetics, which revealed an induced-fit mechanism. The absence of cooperativity produced allosterically activated transitory states displaying velocity against substrate concentration curves with apparent negative cooperativity, due to the simultaneous presence of subunits with different substrate affinities. Reaction kinetics experiments performed at different tertiary conformational relaxation times also reveal the sequential nature of the allosteric activation. We assumed as a minimal model the existence of two tertiary states, t and r, of low and high affinity, respectively, for the substrate and the activator. By fitting the velocity-substrate curves as a linear combination of two hyperbolic functions with K _t and K _r as K_M values, we obtained comparable values to those reported for the quaternary conformers in solution fitted to MWC model. These results are discussed in the background of the known crystallographic structures of T and R EcGNPDA conformers. These results are consistent with the postulates of the Tertiary Two-States (TTS) model.     

Structural Basis for Morpheein-type Allosteric Regulation of Escherichia coli Glucosamine-6-phosphate Synthase: EQUILIBRIUM BETWEEN INACTIVE HEXAMER AND ACTIVE DIMER*

The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.     

2型猪链球菌葡萄糖胺-6-磷酸脱氨酶基因的克隆表达及酶活性测定

目的:克隆表达2型猪链球菌葡萄糖胺-6-磷酸脱氨酶(NagB)编码基因,并测定其酶反应体系活性。方法:根据2型猪链球菌05ZYH33基因组序列,全合成nagB基因(ssu05_0195),并将其克隆至pET32a载体,在大肠杆菌中表达;利用Ni亲和层析柱纯化表达产物,获得纯化的NagB蛋白,Western印迹鉴定后测定其酶反应体系活性。结果:在大肠杆菌中高效表达了nagB基因,重组NagB相对分子质量约为56×10~3;在25℃、pH9.5、底物浓度为15 mmol/L、反应40 min时,NagB酶促反应体系表现出最大活性;在最适条件下,2型猪链球菌中NagB酶促反应体系的体外活性为3.73 U/mL,酶比活为12.43 U/mg。结论:2型猪链球菌05ZYH33中含有编码NagB的nagB基因,在原核系统中表达的NagB蛋白具有酶学活性。     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

Genetic Mapping of Loci for Glucose-6-Phosphate Dehydrogenase, Gluconate-6-Phosphate Dehydrogenase, and Gluconate-6-Phosphate Dehydrase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting the constitutive enzymes glucose-6-phosphate dehydrogenase (zwf) and gluconate-6-phosphate dehydrogenase (gnd), and the inducible enzyme gluconate-6-phosphate dehydrase (edd), were determined by conjugation and transduction experiments, chiefly by three-factor crosses. They are in the same region of the chromosome, and their order is gnd-his-(edd, zwf)-aroD; gnd and his are cotransduceable, as are zwf and edd. The position of gnd in Salmonella typhimurium was shown to be similar to that in E. coli.     

Isolation of Specialized Transducing Bacteriophages for Gluconate 6-Phosphate Dehydrogenase (gnd) of Escherichia coli

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.     

2-Keto-3-Deoxygluconate 6-Phosphate Aldolase Mutants of Escherichia coli

A new mutation in Escherichia coli, giving inability to grow on gluconic, glucuronic, or galacturonic acids, has been identified as complete deficiency of 2-keto-3-deoxygluconate 6-phosphate (KDGP) aldolase activity. The genetic map position of the locus, eda, is about 35 min. The inability to grow on the uronic acids was expected, because the aldolase is on the sole known pathway of their metabolism. However, inability to grow on gluconate was less expected, because the hexose monophosphate shunt might be used, as happens in mutants blocked in the previous step, edd, of the Entner-Doudoroff pathway. The likely explanation of gluconate negativity is inhibition by accumulated KDGP, because gluconate is inhibitory to growth on other substances, and one type of gluconate revertant is eda(-), edd(-). KDGP is probably the inducer of KDGP aldolase.     

Induction of Adenosine Deaminase in Escherichia coli

Supplementing the salts-glucose medium of Escherichia coli with adenine initiates induction of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), growth inhibition, and an increased potential for the net deamination of adenine. The extent and duration of these events are proportional to the initial adenine concentration and are dependent upon adenylate pyrophosphorylase and repression of histidine biosynthesis for maximal expression. The conversion of adenine to hypoxanthine, though limited in rate, occurs concurrently with induction and accounts for the progressively decreasing rate of deaminase induction, since hypoxanthine is a relatively ineffective inducer. The subsequent decrease in deaminase activity is due to dilution by continued cell division and by enzyme inactivation which occurs during the late-log and early-stationary phases. The partially purified deaminase is labile to a number of environmental conditions, particularly to phosphate buffers of pH 6.8 or less. A disproportionately slow rate of adenine deamination by cells utilizing lactate permits a more prolonged period of induction and, consequently, a greater quantity of enzyme to be synthesized; cell division, but not enzyme inactivation, reduces enzyme concentration. The adenosine deaminases of Aerobacter aerogenes and Salmonella typhimurium are not inducible.     

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Effects of Phosphonic Acid Analogues of Glycerol-3-Phosphate on the Growth of Escherichia coli

The four-carbon phosphonate, 3,4-dihydroxybutyl-1-phosphonate, is similar to glycerol-3-phosphate in its ability to inhibit cell growth of Escherichia coli strain 8 cultured in low-phosphate synthetic medium supplemented with either succinate or casein hydrolysate as the sole carbon source. The three-carbon phosphonate, 2,3-dihydroxypropyl-1-phosphonate, does not appear to exhibit a similar effect. The inhibition caused by the four-carbon phosphonate differs from that caused by glycerol-3-phosphate in at least three ways. (i) Its inhibitory effect is not offset by the presence of glucose in the culture medium. (ii) It is capable of exerting its inhibitory effect on cells containing an active aerobic glycerol-3-phosphate dehydrogenase. (iii) Its inhibitory effect is maintained in synthetic medium containing high concentrations of inorganic phosphate. The four-carbon phosphonate appears to be bacteriostatic and inhibits the uptake of labeled glycerol-3-phosphate by E. coli strain 8.     

Chromosomal Location of a Gene for Fructose 6-Phosphate Kinase in Escherichia coli

Pfk lies between rha and glpK.     

Studies with a Gluconate-6-Phosphate Dehydrogenase Mutant in Escherichia coli Strain C

A map location of the gluconate-6-phosphate dehydrogenase (gnd) marker was estimated in Escherichia coli C at approximately 46 min by P1 transduction. The gnd locus appears to lie between the co-transducible histidine and prophage P2 location I markers.     

Kinetics of Exogenous Induction of the Hexose-6-Phosphate Transport System of Escherichia coli

The kinetics of the exogenous induction of the hexose-phosphate transport system by glucose-6-phosphate (G6P) was investigated. The induction of this system by extracellular but not intracellular G6P was confirmed. The differential rate of synthesis was linear, a function of the extracellular concentration of G6P and independent of the previous induction history of the culture. Neither maintenance nor autocatalysis, phenomena described in the induction of the lac operon, were observed in the exogenous induction of hexose-phosphate transport. Fructose-6-phosphate, a potent competitive inhibitor of G6P influx, had no effect on the induction of the system by G6P, indicating that the transport of inducer was not involved in the induction process.     

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

Regulation by Membrane Fluidity of the Allosteric Behavior of the (Ca2+)-Adenosine Triphosphatase from Escherichia coli

The allosteric properties of the membrane-bound (Ca(2+))-adenosine triphosphatase of an unsaturated fatty acid auxotroph of Escherichia coli were studied in membranes with different fatty acid compositions. The Hill coefficient of the inhibition by Na(+) ranged from 1.4, in the case where the auxotroph was grown with cis-vaccenic acid as supplement, to 2.8 when grown on linolenic acid. The results indicate that no fatty acid is particularly involved in the allosteric phenomena. A correlation between the values of the Hill coefficient and the double bond index or the ratio of the double bond index saturated to the fatty acids of the membrane was found. These facts are interpreted as a modulation by the membrane fluidity of the allosteric behavior of the membrane-bound enzyme. The general biological character of this phenomenon is discussed in this paper.     

Allosteric transition and substrate binding are entropy-driven in glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6P-deaminase (EC 3.5.99.6, formerly glucosamine-6-phosphate isomerase, EC 5.3.1.10) from Escherichia coli is an attractive experimental model for the study of allosteric transitions because it is both kinetically and structurally well-known, and follows rapid equilibrium random kinetics, so that the kinetic K(m) values are true thermodynamic equilibrium constants. The enzyme is a typical allosteric K-system activated by N-acetylglucosamine 6-P and displays an allosteric behavior that can be well described by the Monod-Wyman-Changeux model. This thermodynamic study based on the temperature dependence of allosteric parameters derived from this model shows that substrate binding and allosteric transition are both entropy-driven processes in E. coli GlcN6P deaminase. The analysis of this result in the light of the crystallographic structure of the enzyme implicates the active-site lid as the structural motif that could contribute significantly to this entropic component of the allosteric transition because of the remarkable change in its crystallographic B factors.