Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site

The active site of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been probed using site-directed mutagenesis and inhibitor binding techniques. Replacement of a specific glycyl with an alanyl or a prolyl with a seryl residue in a highly conserved region confers glyphosate tolerance to several bacterial and plant EPSPS enzymes, suggesting a high degree of structural conservation between these enzymes. The glycine to alanine substitution corresponding to Escherichia coli EPSPS G96A increases the Ki(app) (glyphosate) of petunia EPSPS 5000-fold while increasing the Km(app)(phosphoenolpyruvate) about 40-fold. Substitution of this glycine with serine, however, abolishes EPSPS activity but results in the elicitation of a novel EPSP hydrolase activity whereby EPSP is converted to shikimate 3-phosphate and pyruvate. This highly conserved region is critical for the interaction of the phosphate moiety of phosphoenolpyruvate with EPSPS.     

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.     

Biotin synthase: insights into radical-mediated carbon-sulfur bond formation

The enzyme cofactor and essential vitamin biotin is biosynthesized in bacteria, fungi, and plants through a pathway that culminates with the addition of a sulfur atom to generate the five-membered thiophane ring. The immediate precursor, dethiobiotin, has methylene and methyl groups at the C6 and C9 positions, respectively, and formation of a thioether bridging these carbon atoms requires cleavage of unactivated CH bonds. Biotin synthase is an S-adenosyl-l-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals, high-energy carbon radicals that can directly abstract hydrogen atoms from dethiobiotin. The available experimental and structural data suggest that a [2Fe-2S](2+) cluster bound deep within biotin synthase provides a sulfur atom that is added to dethiobiotin in a stepwise reaction, first at the C9 position to generate 9-mercaptodethiobiotin, and then at the C6 position to close the thiophane ring. The formation of sulfur-containing biomolecules through a radical reaction involving an iron-sulfur cluster is an unprecedented reaction in biochemistry; however, recent enzyme discoveries suggest that radical sulfur insertion reactions may be a distinct subgroup within the burgeoning Radical SAM superfamily. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Involvement of lysine-193 of the conserved "K-T-G-G" motif in the catalysis of maize starch synthase IIa

It has been suggested that the lysine residue in the conserved K-T-G-G motif could be the substrate ADP-glucose binding site of Escherichia coli glycogen synthase (GS). Since the K-X-G-G motif is highly conserved between E. coli GS and all the maize starch synthase (SS) isozymes, it has become widely accepted that the lysine in the conserved K-T-G-G motif may also function as the ADPGlc binding site of maize SS. We have used chemical modification and site-directed mutagenesis to study the function of lysine residues in SS. Pyridoxal-5'-phosphate inactivated maize SSIIa activity in a time and concentration dependent manner. ADPGlc completely protected SSIIa from inactivation by pyridoxal-5'-phosphate, indicating that lysine residue(s) could be important for ADPGlc binding and enzyme catalysis. In contrast to E. coli GS, mutation of conserved lysine193 (K-T-G-G) in maize SS did not alter the ADPGlc binding while significantly changing the enzyme activity toward different primers. Our results suggest that lysine-193 (K-T-G-G) is not directly involved in ADPGlc binding, instead mutation in the conserved lysine position affected the primer preference.     

Site-directed mutagenesis of the conserved beta subunit tyrosine 331 of Escherichia coli ATP synthase yields catalytically active enzymes

The ATP synthases of eubacteria and eukaryotes possess a conserved tyrosine (beta 331) that is labeled by ATP analogs and is believed to be at the catalytic site. In this report, this tyrosine was replaced by Phe, Ser, Cys, Gly, and Ala in an attempt to determine its role in catalysis. Each of the beta 331 mutant strains assembled an ATP synthase. Membranes from the beta 331-Ser, -Cys, -Ala, or -Gly strains showed strongly attenuated ATP hydrolysis and ATP-driven proton-pumping activities. The beta 331-Phe membranes showed nearly normal ATPase and functional proton pumping. A new purification procedure yielding highly active unc+ F1 (ATPase rates greater than 1000 s-1) allowed rapid isolation of soluble F1-ATPases. Kinetic analyses of purified enzymes confirmed that the structural and functional properties of beta 331-Tyr can be substituted by Phe but not effectively by Ser, Cys, Ala, or Gly. Since all of the beta 331 mutant enzymes catalyzed measurable ATP hydrolysis, it is clear that beta 331-Tyr is not directly involved in the bond making-breaking steps of catalysis. The ability of the beta 331-Phe enzyme to rapidly bind and hydrolyze ATP, and the results with other beta 331 mutant enzymes, suggests that a residue with an aromatic character is required at this position.     

Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.     

PCNA binding through a conserved motif

Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of several proteins. The cell cycle regulatory protein, p21, and the replication endonuclease, Fen1, have already been described as competing for PCNA binding. Two recent reports have identified DNA (cytosine-5)methyltransferase (MCMT) and the DNA repair endonuclease XPG as binding to PCNA.^1,2 The remarkable thing about these interactions is that they all seem to occur through a conserved motif that is likely to contact the same site on PCNA. This has fascinating implications for a regulatory network linking these diverse protein functions. BioEssays 20 :195–199, 1998. © 1998 John Wiley & Sons, Inc.     

Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.     

Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity.

Comparison of the farnesyl diphosphate (FPP) synthase amino acid sequences from four species with amino acid sequences from the related enzymes hexaprenyl diphosphate synthase and geranylgeranyl diphosphate synthase show the presence of two aspartate rich highly conserved domains. The aspartate motif ((I, L, or V)XDDXXD) of the second of those domains has homology with at least 9 prenyl transfer enzymes that utilize an allylic prenyl diphosphate as one substrate. In order to investigate the role of this second aspartate-rich domain in rat FPP synthase, we mutated the first or third aspartate to glutamate, expressed the wild-type and mutant enzymes in Escherichia coli, and purified them to apparent homogeneity using a single chromatographic step. Approximately 12 mg of homogeneous protein was isolated from 120 mg of crude bacterial extract. The kinetic parameters of the purified wild-type recombinant FPP synthase containing the DDYLD motif were as follows: Vmax = 0.84 mumol/min/mg; GPP Km = 1.0 microM; isopentenyl diphosphate (IPP) Km = 2.7 microM. Substitution of glutamate for the first aspartate (EDYLD) decreased the Vmax by over 90-fold. The Km for IPP increased, whereas the Km for GPP remained the same in this D243E mutant. Substitution of glutamate for the third aspartate (DDYLE) did not result in altered enzyme kinetics in the D247E mutant. These results suggest that the first aspartate in the second domain is involved in the catalysis by FPP synthase.     

Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase

Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine. This activity is dependent on pyridoxal 5'-phosphate (PLP) and dithiothreitol and proceeds through a protein-bound persulfide. Like other cysteine desulfurases, BioB binds 1 equiv of PLP. By site-directed mutagenesis, two conserved cysteines, Cys97 and Cys128, are shown to be critical for cysteine desulfuration and are good candidates as the site for a persulfide. Since biotin synthase activity is greatly increased by PLP and cysteine, even though it does not exceed 1 nmol of biotin/nmol of monomer, it is proposed that cysteine desulfuration is intimately linked to biotin synthesis. New scenarios for sulfur insertion into dethiobiotin, in which cysteine persulfides play a key role, are discussed.     

The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase

The supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in the inner mitochondrial membrane, whose masses are higher than those of ATP synthase dimers.     

The DHHC domain: A new highly conserved cysteine-rich motif

A unique clone from a human pancreatic cDNA library was isolated and sequenced. Examination of the deduced polypeptide sequence of the clone showed a new form of cysteine-rich domain that included a region with the form of a Cys4 zinc-finger-like metal binding site followed by a complex Cys-His region. Searches of the Swiss-Protein data bank found a similar 48-residue domain in fifteen open reading frames deduced from A. thaliana, C. elegans, S. cerevisiae and S. pombe genomic sequences. The high degree of conservation of this domain (13 absolutely conserved and 17 highly conserved positions) suggests that it has an important function in the cell, possibly related to protein-protein or protein-DNA interactions. The gene recognized by the clone is is localized to human chromosome 16, and is conserved in vertebrates. The 2 Kb message is expressed in various human fetal and adult tissues. An antibody made to a peptide sequence of the deduced protein showed reactivity in immunoblots of monkey lung and retinal subcellular fractions and immunohistochemically in late fetal mouse tissues and a limited number of adult mouse tissues, including pancreatic islets, Leydig cells of the testis, and the plexiform layers of the retina.     

A conserved helix-unfolding motif in the naturally unfolded proteins

Among the naturally unfolded proteins there are many polypeptides that retain an extended conformation in the absence of any apparent signal. Using sequence alignment and secondary structure prediction tools, a conserved (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is uncovered in the vicinity of the N-terminus of their unfolded helices. A comparison of these data with published observations allows one to propose that the (LS/SL)(D/E)(D/E)(D/E)X(E/D) motif is a helix-unfolding signal. Furthermore, the strong similarity between this motif and the STXXDE casein kinase II phosphorylation site suggests a regulatory mechanism for the naturally unfolded proteins within the cell.     

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.     

Cold-active citrate synthase: mutagenesis of active-site residues.

A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.     

Saturation site-directed mutagenesis of thymidylate synthase

We have subjected 12 different codons of a synthetic Lactobacillus casei thymidylate synthase (TS) gene to saturation site-directed mutagenesis to create amino acid "replacement sets" at each of those positions. The target residues were chosen because they are highly conserved and because they are important for the structure and function of the protein as indicated by solution and structural studies. The mutagenesis procedure involved excision of a fragment of the synthetic gene containing the target codon, followed by its replacement with a mixture of oligonucleotides which code for all 20 amino acids and the amber stop codon. TS mutants were identified by DNA sequencing, and catalytically active mutants were identified by genetic complementation using a Thy- strain of Escherichia coli. Only 3 of the 12 target amino acids examined were essential for TS activity; and of the 125 total mutants identified, 57 were catalytically active. These results point to a high degree of plasticity of TS in accommodating function with structural change.