The nucleotide sequence of the aroF gene of Escherichia coli and the amino acid sequence of the encoded protein, the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

The translated sequence of aroF, the first structural gene of the tyrosine operon of Escherichia coli, has been determined. The 1068 nucleotides encode the 356 amino acids that form the subunit of the dimeric tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The primary structure of this enzyme has been confirmed by automated Edman degradation of peptide fragments produced by cleavage with cyanogen bromide, limited trypsin digestion, Staphylococcus aureus strain V8 protease, or mild acid hydrolysis. The amino acid sequence of this enzyme is compared with the sequence of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, deduced from the aroG DNA sequence (Davies, W. D., and Davidson, B. E. (1982) Nucleic Acids Res. 10, 4045-4058).     

Identification of amino acid residues contributing to the mechanism of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

L-Serine inhibits the catalytic activity of Escherichia coli D-3-phosphoglycerate dehydrogenase (PGDH) by binding to its regulatory domain. This domain is a member of the ACT domain family of regulatory domains that are modulated by small molecules. A comparison of the phi and psi torsional angle differences between the crystal structures of PGDH solved in the presence and in the absence of L-serine demonstrated a clustering of significant angle deviations in the regulatory domain. A similar clustering was not observed in either of the other two structural domains of PGDH. In addition, significant differences were also observed at the active site and in the Trp-139 loop. To determine if these residues were functionally significant and not just due to other factors such as crystal packing, mutagenic analysis of these residues was performed. Not unexpectedly, this analysis showed that residues that affected the kcat/Km were grouped around the active site and those that affected the serine sensitivity were grouped in the regulatory domain. However, more significantly, residues that affected the cooperativity of inhibition of activity were identified at both locations. These latter residues represent structural elements that participate in both the initial and the ultimate events of the transfer of cooperative behavior from the regulatory domain to the active site. As such, their identification will assist in the elucidation of the pathway of cooperative interaction in this enzyme as well as in the elucidation of the regulatory mechanism of the ACT domain in general.     

Amino acid residue mutations uncouple cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli contains two Gly-Gly sequences that occur at junctions between domains. A previous study (Grant, G. A., Xu, X. L., and Hu, Z. (2000) Biochemistry 39, 7316-7319) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains. Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the K(m) and k(cat). Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domains, which form the active site cleft, have a significant effect on the k(cat) of the enzyme without substantially altering the enzyme's sensitivity to serine. In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition. Interestingly, even though cooperativity of inhibition can be reduced significantly, there is little apparent effect on the cooperativity of serine binding itself. An additional mutant, G336V,G337V, also reduces the cooperativity of inhibition, but in this case serine binding also is reduced to the point at which it cannot be measured by equilibrium dialysis. The double mutant G294V,G336V demonstrates that strain imposed by mutation at one hinge can be relieved partially by mutation at the other hinge, demonstrating linkage between the two hinge regions. These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled. Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme. A comparison of these values with the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme, which will allow modeling of the dynamics of domain movement.     

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.     

Hybrid tetramers reveal elements of cooperativity in Escherichia coli D-3-phosphoglycerate dehydrogenase

d-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when l-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by approximately 58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.     

大肠杆菌磷酸甘油酸脱氢酶突变体的构建及抗反馈抑制效应

磷酸甘油酸脱氢酶(D-3-phosphoglycerate dehydrogenase,PGDH,EC 1.1.1.95)为L-丝氨酸合成途径的关键酶,其编码基因为ser A,其活性受到合成产物L-丝氨酸的反馈抑制调控。为解除丝氨酸的反馈抑制,采用定点突变技术把编码PGDH酶344位组氨酸或346位天冬氨酸或364位天冬氨酸的密码子定点突变为丙氨酸密码子。改造后的ser AFbr被连到表达载体pT7-7上,并转入大肠杆菌Escherichia coli BL21(DE3)中进行表达,破壁回收粗酶液,通过DEAE阴离子柱纯化PGDH突变体,并对其酶活性和IC_(50)值进行了测定。结果,野生型PGDH酶IC_(50)值为7μmol/L,而PGDH双突变体N346A/H344A催化活性与野生型相近,在丝氨酸浓度为160 mmol/L时,其酶活仍保持未添加丝氨酸时酶活的96%,基本解除反馈抑制。     

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.     

Aromatic amino acid aminotransferase of Escherichia coli: nucleotide sequence of the tyrB gene

The tyrB gene of E. coli K-12, which encodes aromatic amino acid aminotransferase (EC 2.6.1.57) was cloned. The nucleotide sequence of about 2 kilobase pairs containing the gene was determined. The coding region of the tyrB gene and the deduced amino acid sequence revealed that the aromatic amino acid aminotransferase of E. coli is homologous with the aspartate aminotransferase.     

The effect of hinge mutations on effector binding and domain rotation in Escherichia coli D-3-phosphoglycerate dehydrogenase

D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli contains two Gly-Gly sequences that have been shown previously to have the characteristics of hinge regions. One of these, Gly(336)-Gly(337), is found in the loop between the substrate binding domain and the regulatory domain. Changing these glycine residues to valine affected the sensitivity of the enzyme to inhibition by L-serine but not the extent of inhibition. The decrease in sensitivity was caused primarily by a decrease in the affinity of the enzyme for L-serine. These mutations also affected the domain rotation of the subunits in response to L-serine binding. A major conclusion of this study was that it defines a minimal limit on the necessary conformational changes leading to inhibition of enzyme activity. That is, some of the conformational differences seen in the native enzyme upon L-serine binding are not critical for inhibition, whereas others are maintained and may play important roles in inhibition and cooperativity. The structure of G336V demonstrates that the minimal effect of L-serine binding leading to inhibition of enzyme activity requires a domain rotation of approximately only 6 degrees in just two of the four subunits of the enzyme that are oriented diagonally across from each other in the tetramer. Moreover the structures show that both pairs of Asn190 to Asn190 hydrogen bonds across the subunit interfaces are necessary for activity. These observations are consistent with the half-the-sites activity, flip-flop mechanism proposed for this and other similar enzymes and suggest that the Asn190 hydrogen bonds may function in the conformational transition between alternate half-the-site active forms of the enzyme.     

Role of an interdomain Gly-Gly sequence at the regulatory-substrate domain interface in the regulation of Escherichia coli. D-3-phosphoglycerate dehydrogenase

The regulatory and substrate binding domains of D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) from Escherichia coli are connected by a single polypeptide strand that contains a Gly-Gly sequence approximately midway between the domains. The potential flexibility of this sequence and its strategic location between major domain structures suggests that it may function in the conformational change leading from effector binding to inhibition of the active site. Site-directed mutagenesis of this region (Gly-336-Gly-337) supports this hypothesis. When bulky side chains were substituted for the glycines at these positions, substantial changes in the ability of serine to inhibit the enzyme were seen with little effect on the activity of the enzyme. The effect of these substitutions could be alleviated by placing a new glycine residue at position 335, immediately flanking the original glycine pair. On the other hand, substituting a glycine at position 338 revealed a critical role for the side chain of Arg-338. This residue may function in stabilizing the conformation about the Gly-Gly turn, resulting in a specific orientation of the adjacent domains relative to each other. Rotation about the phi or psi bonds of either Gly-336 or Gly-337 would have a profound effect on this orientation. The data are consistent with this as a role for the Gly-Gly sequence between the regulatory and substrate binding domains of PGDH.     

Enhanced expression of the Escherichia coli serA gene in a plasmid vector. Purification, crystallization, and preliminary X-ray data of D-3 phosphoglycerate dehydrogenase

The serA gene of Escherichia coli strain K-12, which codes for the cooperative allosteric enzyme D-3-phosphoglycerate dehydrogenase, was inserted into an inducible expression vector which produced phosphoglycerate dehydrogenase as 8% of the soluble protein of E. coli. The purified protein was used to grow several different single crystal forms. One of these, with space group P2(1), appears to contain all four subunits of the tetrameric enzyme in the asymmetric unit and diffracts to sufficient resolution to allow determination of the structure of phosphoglycerate dehydrogenase.     

Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding

The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules. An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown. This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect. In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range. Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH. This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate. Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system. Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding. Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.     

Complete nucleotide sequence of the glutamate dehydrogenase gene from Escherichia coli K-12

A 2.3-kb PstI-ClaI chromosomal DNA segment, carrying the complete coding region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, has been sequenced. The complete amino acid sequence (447 residues) of the GDH monomer has been deduced, and comparisons are made with reported amino acid sequences of GDH from other organisms.     

A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase

Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.