Fluorescent properties of the Escherichia coli D-xylose isomerase active site.

The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.     

Role of the electrostatic loop of Cu,Zn superoxide dismutase in the copper uptake process.

Cu,Zn superoxide dismutases are characterized by the presence of four highly conserved charged residues (Lys120, Glu/Asp130, Glu131 and Lys134), which are placed at the edge of the active site channel and have been shown to be individually involved in the electrostatic attraction of the substrate toward the catalytically active copper ion. By genetic engineering we mutated these four residues into neutrally charged ones (Leu120, Gln130, Gln131, Thr134). The effects of these mutations on the rate of superoxide dismutation were not dramatic. In fact, at two different pH and ionic strength values, the mutant enzyme had a catalytic constant even higher with respect to the wild-type protein, showing that electrostatic interaction at these surface sites is not essential for high catalytic efficiency of the enzyme. The mutant and the wild-type enzyme showed the same degree of inhibition by CN(-), and both were not affected by I(-), showing that mutations did not alter the sensitivity of the enzyme to anions. On the other hand, reconstitution of active enzyme from either the wild-type or mutant copper-free enzymes with a copper(I)-glutathione [Cu(I)-GSH] complex showed that metal uptake by the mutant was much slower than by the wild-type enzyme. The demonstration that the 'electrostatic loop' is apparently conserved to assure optimal copper uptake by the enzyme, rather than fast dismutation, may provide further support to the idea that Cu,Zn superoxide dismutase is a bifunctional protein, acting in cellular defense against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

The contribution of acidic residues to the conformational stability of common-type acylphosphatase

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.     

Mutational analysis ofN-linked glycosylation of esterase 6 inDrosophila melanogaster

The primary sequence of the esterase 6 (EST6) enzyme ofDrosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln byin vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain ofD. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.     

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppA(Tk)), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. DeltaN54SppA(Tk), a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser(162) as the nucleophilic serine and that of Lys(214) as the general base, comprising a Ser/Lys catalytic dyad in SppA(Tk). Kinetic analyses indicated that Ser(184), His(191) Lys(209), Asp(215), and Arg(221) supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser(128) and Tyr(165) not only increased activity levels but also broadened the substrate specificity of SppA(Tk), suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.     

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.     

An innovative application of the "flexible" GRID/PCA computational method: study of differences in selectivity between PGAs from Escherichia coli and a Providentia rettgeri mutant

The original GRID/PCA technique was adapted for the development of a tool potentially useful for the plan of a research strategy in rational enzyme design. The use of the MOVE directive of GRID made it possible to partially take into account protein flexibility, and the multivariate analysis was used as an instrument for focusing only on relevant information related to the differences in enzyme substrate selectivities. The comparison of two different penicillin G acylases, from Escherichia coli and from Providentia rettgeri, was used as a case study; these enzymes are very similar and their reported selectivities differ only for a couple of mutations around the active site. The "flexible" GRID/PCA method was able to correctly predict the observed selectivity differences caused not only by mutations of residues of the active site but also by long range effects on substrate selectivity due to sequence mutations on residues not directly involved in substrate recognition.     

Biochemical and electrochemical characterization of two variant human short-chain acyl-CoA dehydrogenases

Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. Two common variants of this enzyme encoding G185S and R147W substitutions have been identified at an increased frequency compared to the general population in patients with a wide variety of clinical problems, but functional studies of the purified mutant enzymes have shown only modestly changed kinetic properties. Moreover, both amino acid residues are located quite far from the catalytic pocket and the essential FAD cofactor. To clarify the potential relationship of these variants to clinical disease, we have further investigated their thermodynamic properties using spectroscopic and electrochemical techniques. Purified R147W hSCAD exhibited almost identical physical and redox properties to wild-type but only half of the specific activity and substrate activation shifts observed in wild-type enzyme. In contrast, the G185S mutant proved to have impairments of both its kinetic and electron transfer properties. Spectroelectrochemical studies reveal that G185S binding to the substrate/product couple produces an enzyme potential shift of only +88 mV, which is not enough to make the reaction thermodynamically favorable. For wild-type hSCAD, this barrier is overcome by a negative shift in the substrate/product couple midpoint potential, but in G185S this activation was not observed. When G185S was substrate bound, the midpoint potential of the enzyme actually shifted more negative. These results provide valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrate that mutations, regardless of their position in the protein structure, can have a large impact on the redox properties of the enzyme.     

Conserved walker A Ser residues in the catalytic sites of P-glycoprotein are critical for catalysis and involved primarily at the transition state step

P-glycoprotein mutants S430A/T and S1073A/T, affecting conserved Walker A Ser residues, were characterized to elucidate molecular roles of the Ser and functioning of the two P-glycoprotein catalytic sites. Results showed the Ser-OH is critical for MgATPase activity and formation of the normal transition state, although not for initial MgATP binding. Mutation to Ala in either catalytic site abolished MgATPase and transition state formation in both sites, whereas Thr mutants had similar MgATPase to wild-type. Trapping of 1 mol of MgADP/mol of P-glycoprotein by vanadate, shown here with pure protein, yielded full inhibition of ATPase. Thus, congruent with previous work, both sites must be intact and must interact for catalysis. Equivalent mutations (Ala or Thr) in the two catalytic sites had identical effects on a wide range of activities, emphasizing that the two catalytic sites function symmetrically. The role of the Ser-OH is to coordinate Mg(2+) in MgATP, but only at the stage of the transition state are its effects tangible. Initial substrate binding is apparently to an "open" catalytic site conformation, where the Ser-OH is dispensable. This changes to a "closed" conformation required to attain the transition state, in which the Ser-OH is a critical ligand. Formation of the latter conformation requires both sites; both sites may provide direct ligands to the transition state.     

Natural mutations in a 2'-5' oligoadenylate synthetase transgene revealed residues essential for enzyme activity

Unlike other RNA polymerases, 2'-5' oligoadenylate synthetases, a family of interferon-induced enzymes, catalyze the formation of 2'-5', not 3'-5', phosphodiester bonds. Moreover, to be active, these proteins require double-stranded RNA as a cofactor. We have been identifying the specific residues of these proteins that impart their novel properties. Here, we report the identity of three such residues that underwent natural mutations in a transgenic mouse line. When deliberately introduced into recombinant proteins, each of these mutations rendered the protein enzymatically inactive. In an effort to understand the roles of these residues in enzyme activity, new mutants carrying other residues in one of these three sites were generated. Detailed characterization of the properties of the mutant proteins revealed that Lys 404 is needed for proper binding of the acceptor substrate, Pro 500 provides structural flexibility to the protein, and Ser 471 is probably required for its proper folding. This study illustrates the power of using natural mutations in transgenes as guides for studying structure-function relationships of proteins.     

HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.

Internal fatty acylation of proteins is a recognized means of modifying biological behavior. Escherichia coli hemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue epsilon-amino groups; HlyC catalyzes the acyl transfer from acyl-acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.     

Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the hydrolysis of beta-(1, 4)-glycosidic bonds in chitin, the major structural polysaccharide present in the cuticle and gut peritrophic matrix of insects. Two conserved regions have been identified from amino acid sequence comparisons of family 18 glycosyl hydrolases, which includes Manduca sexta (tobacco hornworm) chitinase as a member. The second of these regions in M. sexta chitinase contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. In this study the functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins, D142E, D142N, D144E, D144N, E146D and E146Q, as well as the wild-type enzyme, were produced using a baculovirus-insect cell line expression system. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic and substrate binding properties were determined. Circular dichroism spectra of the mutant proteins were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was required for enzymatic activity because mutants E146Q and E146D were devoid of activity. D144E retained most of the enzymatic activity, but D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity relative to the wild-type enzyme. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicate that E146 probably functions as an acid/base catalyst in the hydrolytic mechanism, as do homologous residues in other glycosyl hydrolases. D144 apparently functions as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.     

Rational design of ornithine decarboxylase with high catalytic activity for the production of putrescine

Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes.     

Construction of new ligand binding sites in proteins of known structure. II. Grafting of a buried transition metal binding site into Escherichia coli thioredoxin.

In an accompanying paper a computational procedure is described, which introduces new ligand-binding sites into proteins of known structure. Here we describe the experimental implementation of one of the designs, which is intended to introduce a copper-binding site into Escherichia coli thioredoxin. The new binding site can be introduced with a minimum of four amino acid changes. The binding site is buried so that structural rules for making mutations in the hydrophobic core of a protein, as well as for the introduction of new functions, are being tested in this experiment. The mutant protein is folded even in the absence of metals, and variants that retain the original activity of thioredoxin can be isolated. The protein has gained a metal-binding site specific for transition metals. The metal co-ordination chemistry at the binding site varies depending on the metal that is introduced into it. Mercury(II) is co-ordinated in the expected manner. Copper(II) binds in a way that was not anticipated in the original design. It appears to use two of the four residues intended to form the co-ordination sphere, and two other residues that were not part of the original set of mutations. It is therefore necessary not only to introduce new functional groups to form a new site, but also to consider and remove alternative modes of binding.     

Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.     

Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis

NAD kinase is the only known enzyme catalyzing the formation of NADP, a coenzyme implicated in most reductive biosynthetic reactions and in many antioxidant defense systems. Despite its importance, nothing is known regarding its structure or mechanism of catalysis. Mycobacterium tuberculosis NAD kinase has been overexpressed in Escherichia coli and purified to homogeneity. The molecular and kinetic properties of the enzyme resulted in significant differences from those reported by others on a proteolytically degraded form of the protein. Indeed the full-length enzyme displays an allosteric behavior and shows a strict preference for inorganic polyphosphate as the phosphate donor. It is inhibited by the reaction product NADP and by both NADH and NADPH. The mycobacterial enzyme shares with all other known NAD kinases a highly conserved region (spanning residues 189-210), particularly rich in glycines, which differs from the primary sequences of all previously identified nucleotide-binding sites. Alanine-scanning mutagenesis performed on 11 conserved residues within this domain revealed its importance in catalysis. A total of 6 of 11 mutated proteins completely lost the enzymatic activity while retaining the same oligomeric state of the wild-type protein, as demonstrated by gel-filtration analysis. Substitutions of S199 and G208 with alanine rendered enzyme versions with reduced activity. Their kinetic characterization, performed on purified proteins, revealed kinetic parameters toward ATP and polyphosphate similar to those of the wild-type enzyme. On the contrary, when the kinetic analysis was performed by using NAD as the variable substrate, significant differences were observed with respect to both the allosteric behavior and the catalytic efficiency, suggesting that the mutated region is likely involved in NAD binding.     

Segmental motion in catalysis: investigation of a hydrogen bond critical for loop closure in the reaction of triosephosphate isomerase.

A residue essential for proper closure of the active-site loop in the reaction catalyzed by triosephosphate isomerase is tyrosine-208, the hydroxyl group of which forms a hydrogen bond with the amide nitrogen of alanine-176, a component of the loop. Both residues are conserved, and mutagenesis of the tyrosine to phenylalanine results in a 2000-fold drop in the catalytic activity (kcat/Km) of the enzyme compared to the wild-type isomerase. The nature of the closure process has been elucidated from both viscosity dependence and primary isotope effects. The reaction catalyzed by the mutant enzyme shows a viscosity dependence using glycerol as the viscosogen. This dependence can be attributed to the rate-limiting motion of the active-site loop between the "open" and the "closed" conformations. Furthermore, a large primary isotope effect is observed with [1-2H]dihydroxyacetone phosphate as substrate [(kcat/Km)H/(kcat/Km)D = 6 +/- 1]. The range of isotopic experiments that were earlier used to delineate the energetics of the wild-type isomerase has provided the free energy profile of the mutant enzyme. Comparison of the energetics of the wild-type and mutant enzymes shows that only the transition states flanking the enediol intermediate have been substantially affected. The results suggest either that loop closure and deprotonation are coupled and occur in the same rate-limiting step or that these two processes happen sequentially but interdependently. This finding is consistent with structural information that indicates that the catalytic base glutamate-165 moves 2 A toward the substrate upon loop closure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion

Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.     

Probing the role of the C-terminus of Bacillus subtilis chorismate mutase by a novel random protein-termination strategy

A novel strategy combining random protein truncation and genetic selection has been developed to identify dispensable C-terminal segments of an enzyme. This approach, which entails the random introduction of premature termination codons, was applied to the last 17 residues of chorismate mutase from Bacillus subtilis (BsCM). Although structurally ill-defined, the C-terminus of BsCM has been proposed to cap the active site upon substrate binding and affect catalysis. However, sequence patterns of 178 selected gene variants show that the final 11 residues of the protein can be mutated and even removed without significantly impairing activity in vivo. In fact, none of the randomized residues is absolutely required, but a preference for wild-type Lys111, Ala112, Leu115, and Arg116 is apparent. These residues are part of a C-terminal 3(10)-helix and provide contacts with the rest of the protein or its ligands. The kinetic parameters of selected enzyme variants show that truncations and mutations do not significantly impair catalytic turnover (k(cat)) but substantially decrease k(cat)/K(m). Thus, while the 17 C-terminal residues of BsCM do not participate directly in the chemical rearrangement, they appear to contribute to enzymatic efficiency via uniform binding of the substrate and transition state.