Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.     

Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.     

Molecular basis of exopeptidase activity in the C-terminal domain of human angiotensin I-converting enzyme: insights into the origins of its exopeptidase activity

Proteolytic processing is a primary means of biological control. Exopeptidases use terminal anchoring interactions to restrict cleavage at peptide substrate N or C termini. In contrast, internal peptide bond targeting by endopeptidases is through context-driven recognition. Angiotensin I-converting enzyme (ACE), a zinc metalloproteinase, has tandem duplicate catalytic domains, N- and C-terminal, each of which is a dual specificity enzyme with exo- and endocarboxypeptidase activities. The mechanisms by which ACE evolved from its endopeptidase ancestors as a dual specificity enzyme have not been defined. Based on kinetic studies of wild-type and mutant forms of the C-terminal catalytic domain of human ACE and of the ACE substrates angiotensin I, substance P, and bradykinin, as well as considerations of the ACE x-ray structure, we provide evidence that the acquisition of its exopeptidase activity is due to novel evolutionary specializations. These involve not only interactions between the S(2)' subsite cognate for the C-terminal substrate P(2)' side chain, acting in concert with carboxylate-docking interactions with Lys(1087) and Tyr(1096), but also electrostatic selection against a cationic C-terminal substrate carboxylate. With a blocked C terminus, substrate side chain interactions are dominant in cleavage site selection. In the evolution of obligate exopeptidases from endopeptidase ancestors, mutations that destroy context-driven peptide bond targeting are likely to have followed the acquisition of terminal docking interactions. Evolutionary intermediates between endopeptidases and obligate exopeptidases could therefore have been dual specificity proteinases like ACE.     

Probing the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) by mutational analysis

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.     

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.     

Multiple paths for nonphysiological transport of K+ in Escherichia coli

Mutants of Escherichia coli lacking all of the known saturable K+ transport systems, "triple mutants," require elevated K+ concentrations for growth. K+ transport activity in such mutants, called TrkF activity, has low substrate specificity and a low rate that increases with increasing external pH. Attempts to isolate mutants requiring even higher concentrations of K+ failed, implying that either TrkF is essential or is composed of multiple minor K+ transport activities. Instead, we sought mutations that allowed triple mutants to grow at lower K+ concentrations. Mutations so identified include ones altering MscL, the large mechanosensitive channel, or Opp, the oligopeptide permease. However, a possible contribution of wild-type Opp and MscL to TrkF activity was not proven. In contrast, expression of wild-type ProP, TrkG, and TrkH proteins increased uptake when encoded on multicopy plasmids. In all of these situations, the driving force for K+ appeared to be the transmembrane electric potential, and in most cases substrate specificity was low; these are characteristics of TrkF activity. These results support the view that TrkF is composed of multiple, "aberrant" K+ transport activities, i.e., paths that, regardless of their physiological function, allow K+ to cross the cell membrane by a uniport process.     

Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K(M) in reactions with the novel para-nitrophenyl-beta-d-fucopyranoside substrate; 39-fold decrease in reactivity with the "native"para-nitrophenyl-beta-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/K(M) in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.     

Design and characterization of new β-glucuronidase active site variants with altered substrate specificity

Objective

To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.

Results

Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-d-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved k_cat/K_M value towards pNP-gal compared to wild-type GUS, up to 9 × 10⁸-fold shift in specificity from p-nitrophenyl-β-d-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 10³-fold increase in specificity shift compared to a previously evolved GUS variant.

Conclusions

The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.

     

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.     

The Amino Acid Sequence of Ribitol Dehydrogenase-F, a Mutant Enzyme with Improved Xylitol Dehydrogenase Activity

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 P196 mutation that would disrupt a surface -helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.     

Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.     

Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase

Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.     

Familial Alzheimer disease presenilin-1 mutations alter the active site conformation of γ-secretase

Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.     

Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme.

Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L. Wild-type and mutant proteins were purified after synthesis in Escherichia coli. These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra. Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme. While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP. All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme. Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2. The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits.     

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.     

The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k(red) (the limiting rate of enzyme reduction by substrate at high [S]) decreased approximately 20-fold in an R310K variant and 2 x 10(4)-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed approximately 4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k(red) greater than 10(3)), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k(red) less than 50-fold). The effect was such that the 4000-fold range in k(red) seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.     

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.     

A combinatorial approach to substrate discrimination in the P450 CYP1A subfamily

A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through the kinetic analysis of combinatorial variants of increasing complexity. The less complex variants correspond to multiple mutations within a short segment of their sequence. The more complex variants correspond to mosaic P450s recombining 1A1 and 1A2 sequences (up to 5 crossovers per sequence). Fifty-eight such functional CYP1A variants and parental wild-type enzymes were expressed in yeast and assayed with 7-alkoxyresorufins and ethoxyflurorescein ethyl ester as substrates. Observed kinetic data were analyzed by multivariate statistical analyses and hierarchical clustering in order to highlight correlations and identify potential sequence-activity relationships within the three-dimensional function space investigated. Several variants are outliers in these representations and show a redistribution of their substrate specificity compared to wild-type CYP1As. Some combinations of sequence elements were identified that significantly discriminate between 1A1 and 1A2 for these three substrates. The comparison of this combinatorial approach with previous results of site-directed mutagenesis is discussed.