Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues

The flavoenzyme UDP-galactopyranose mutase (UGM) is a mediator of cell wall biosynthesis in many pathogenic microorganisms. UGM catalyzes a unique ring contraction reaction that results in the conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UDP-Galf is an essential precursor to the galactofuranose residues found in many different cell wall glycoconjugates. Due to the important consequences of UGM catalysis, structural and biochemical studies are needed to elucidate the mechanism and identify the key residues involved. Here, we report the results of site-directed mutagenesis studies on the absolutely conserved residues in the putative active site cleft. By generating variants of the UGM from Klebsiella pneumoniae, we have identified two arginine residues that play critical catalytic roles (alanine substitution abolishes detectable activity). These residues also have a profound effect on the binding of a fluorescent UDP derivative that inhibits UGM, suggesting that the Arg variants are defective in their ability to bind substrate. One of the residues, Arg280, is located in the putative active site, but, surprisingly, the structural studies conducted to date suggest that Arg174 is not. Molecular dynamics simulations indicate that closed UGM conformations can be accessed in which this residue contacts the pyrophosphoryl group of the UDP-Gal substrates. These results provide strong evidence that the mobile loop, noted in all the reported crystal structures, must move in order for UGM to bind its UDP-galactose substrate.     

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T-protein.

The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the l-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.     

Cysteine mutagenesis of the amino acid residues of transmembrane helix I in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a sugar-cation cotransport system that utilizes Na(+), Li(+), or H(+). This membrane transport protein consists of 12 transmembrane helices. Starting with the cysteine-less melibiose carrier, cysteine has been substituted individually for amino acids 17-37, which includes all of the residues in membrane helix I. The carriers with cysteine substitutions were studied for their transport activity and the effect of the water soluble sulfhydryl reagent p-chloro- mercuribenzenesulfonic acid (PCMBS). Cysteine substitution caused loss of transport activity in six of the mutants (G17C, K18C, D19C, Y32C, T34C, and D35C). PCMBS caused greater than 50% inhibition in eleven mutants (F20C, A21C, I22C, G23C, I24C, V25C, Y26C, M27C, Y28C, M30C, and Y31C). We suggest that the residues whose cysteine derivatives were inhibited by PCMBS face the aqueous channel and that helix I is completely surrounded by aqueous environment. Second site revertants were isolated from K18C and Y31C. The revertants were found to have mutations in helices I, IV, and VII.     

Site-specific mutagenesis of Escherichia coli asparaginase II. None of the three histidine residues is required for catalysis.

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid beta-hydroxamate or impaired aspartate binding. The relative activities towards L-Asn, L-Gln, and l-aspartic acid beta-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in 1H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. 1H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site.     

Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product.

Fragments of the tyrA gene of Escherichia coli, when suitably engineered, can express either the chorismate mutase activity or the prephenate dehydrogenase activity without the other.     

Metabolic engineering of Escherichia coli for L-tyrosine production by expression of genes coding for the chorismate mutase domain of the native chorismate mutase-prephenate dehydratase and a cyclohexadienyl dehydrogenase from Zymomonas mobilis

The expression of the feedback inhibition-insensitive enzyme cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase domain from native chorismate mutase-prephenate dehydratase (PheA(CM)) from Escherichia coli was compared to the expression of native feedback inhibition-sensitive chorismate mutase-prephenate dehydrogenase (CM-TyrA(p)) with regard to the capacity to produce l-tyrosine in E. coli strains modified to increase the carbon flow to chorismate. Shake flask experiments showed that TyrC increased the yield of l-tyrosine from glucose (Y(l-Tyr/Glc)) by 6.8-fold compared to the yield obtained with CM-TyrA(p). In bioreactor experiments, a strain expressing both TyrC and PheA(CM) produced 3 g/liter of l-tyrosine with a Y(l-Tyr/Glc) of 66 mg/g. These values are 46 and 48% higher than the values for a strain expressing only TyrC. The results show that the feedback inhibition-insensitive enzymes can be employed for strain development as part of a metabolic engineering strategy for l-tyrosine production.     

Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli.

Site-directed mutagenesis has been used to identify a number of charged residues essential for the transport activity of the PheP protein. These residues are highly conserved in the cluster of amino acid transporters. However, some other conserved residues and a number of aromatic residues have been shown not to be essential for transport activity.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Cysteine substitutions for individual residues in helix VI of the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H⁺, Na⁺, or Li⁺. In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22–100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes. Received: 6 March 2001/Revised: 14 May 2001     

Structure-based mutagenesis of SigE verifies the importance of hydrophobic and electrostatic residues in type III chaperone function

Despite sharing little sequence identity, most type III chaperones display a similar homodimeric structure characterized by negative charges distributed broadly over their entire surface, interspersed with hydrophobic patches. Here we have used SigE from Salmonella as a model for class IA type III chaperones to investigate the role of these surface-exposed residues in chaperone function. SigE is essential for the stability, secretion and translocation of its cognate effector, SopB (SigD). We analysed the effect of mutating nine conserved hydrophobic and electronegative surface-exposed amino acids of SigE on SopB binding, stability, secretion and translocation. Six of these mutations affected some aspect of SigE function (Leu14, Asp20, Leu22, Leu23, Ile25 and Asp51) and three were without effect (Leu54, Glu92 and Glu99). Our results highlight that both hydrophobic and electronegative surfaces are required for the function of SigE and provide an important basis for the prediction of side-chain requirements for other chaperone-effector pairs.     

Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin.

The HlyA secretion signal sequence of approximately 46 residues is predicted to contain helix I and an amphipathic helix II separated by a short loop including the conserved Phe residue, F-989. All nine substitutions of Phe-989 drastically reduce secretion of HlyA. Directed mutagenesis identified a functional hot spot, EISK, in helix II. However, genetic analysis did not provide strong support for a functional helix II; rather, the results emphasized that individual residues, for example, E-978 and F-989, are essential irrespective of a specific secondary structure.     

The importance of methionine residues for the catalysis of the biotin enzyme, transcarboxylase. Analysis by site-directed mutagenesis.

Almost all biotin enzymes contain the conserved tetrapeptide Ala-Met-Bct-Met (Bct, N epsilon-biotinyl-L-lysine). In the 1.3 S biotinyl subunit of transcarboxylase (TC), this sequence is present between positions 87 and 90. The conserved nature of these amino acids implies a critical role in the function of biotin enzymes. In order to examine the role of these conserved amino acids, point mutations in the gene encoding the 1.3 S subunit have been made by site-directed mutagenesis to generate A87G, M88L, M90L, M88T, M88C, M88A, and a double mutant A87M, M88A in the 1.3 S subunit. TC, a multisubunit enzyme containing 12 S, 5 S, and 1.3 S subunits, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate (overall reaction). TC can be dissociated into individual subunits and also reconstituted by assembling isolated subunits to a fully active form. The mutants of the 1.3 S subunit have been reconstituted with native 5 S and 12 S subunits from Propionibacterium shermanii. The effects of mutations on the activity of TC were compared with that of TC-1.3 S wild type (WT) prepared in a similar manner. The results show that any substitution of a residue in the conserved tetrapeptide causes impairment of the rate of TC activity. Comparison of gel filtration profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron micrographs of the TC assembled with mutant 1.3 S and with wild type 1.3 S subunits showed that the impairment of the overall activity was not due to a failure of the subunits to assemble into complexes. Steady state kinetic analysis using the mutant 1.3 S subunits indicated that the Km for methylmalonyl-CoA or pyruvate did not change significantly indicating that the binding of substrates is not altered. However, the kcat values were significantly lower for mutants at positions 87 and 88 than for those at position 90. The replacement of methionine at position 88 either by hydrophobic or hydrophilic residues significantly altered the activity in the overall reaction, while similar substitution at position 90 did not dramatically alter the kcat. These results suggest that Ala-87 and Met-88 are catalytically critical in the conserved tetrapeptide.     

Probing the overlap of chorismate mutase and prephenate dehydrogenase sites in the escherichia coli T-protein: a dehydrogenase-selective inhibitor

An inhibitor of prephenate dehydrogenase has been identified that has no effect on the chorismate mutase activity in the Escherichia coli T-protein, thus supporting the idea of two separate active sites.     

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.     

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.     

The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase.

In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine. The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site. However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme. The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site. However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl. 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme. The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes. These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group.     

Induced mutagenesis in dam- mutants of Escherichia coli: a role for 6-methyladenine residues in mutation avoidance.

Summary E. coli strains carrying the dam-3 and dam-4 mutations resulting in reduced levels of 6-methyladenine in the DNA have been found to be more sensitive to base analogue mutagenesis than dam ⁺ strains. Mutagenesis by EMS was also found to be enhanced in dam ^– strains. Dam ^– mutants however were not found to be hypermutable by UV light. It is concluded that the dam ^– strains are deficient in the correct repair of mispairing lesions. The data are consistent with the hypothesis that 6-methyladenine residues in the DNA are involved in strand discrimination during mismatch correction.