Evolution toward Small Molecule Inhibitor Resistance Affects Native Enzyme Function and Stability, Generating Acarbose-insensitive Cyclodextrin Glucanotransferase Variants

Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC50 values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants. Small molecule inhibitors play an essential role in the selective inhibition of enzymes associated with human infection and metabolic disorders. Targeted enzymes may evolve toward inhibitor resistance through selective incorporation of mutations. Acquisition of insensitivity may, however, result in profound devolution of native enzyme function and stability. We therefore investigated the consequential effects on native function and stability by evolving a cyclodextrin glucanotransferase (CGTase) enzyme toward insensitivity to the small molecule inhibitor of the protein, acarbose. Error-prone PCR mutagenesis was applied to search the sequence space of CGTase for acarbose-insensitive variants. Our results show that all selected mutations were localized around the active site of the enzyme, and in particular, at the acceptor substrate binding sites, highlighting the regions importance in acarbose inhibition. Single mutations conferring increased resistance, K232E, F283L, and A230V, raised IC50 values for acarbose between 3,500- and 6,700-fold when compared with wild-type CGTase but at a significant cost to catalytic efficiency. In addition, the thermostability of these variants was significantly lowered. These results reveal not only the relative ease by which resistance may be acquired to small molecule inhibitors but also the considerable cost incurred to native enzyme function and stability, highlighting the subsequent constraints in the further evolutionary potential of inhibitor-resistant variants. Many small molecule inhibitors play a central role in current treatment of human diseases, targeting an essential structure or process of the bacterium, virus, or host cell itself. Bactericidal antibiotics including aminoglycosides, macrolides, and tetracyclines are all potent inhibitors of bacterial protein synthesis (1Jana S. Deb J.K. Appl. Microbiol. Biotechnol. 2006; 70: 140-150Crossref PubMed Scopus (224) Google Scholar, 2Magnet S. Blanchard J.S. Chem. Rev. 2005; 105: 477-498Crossref PubMed Scopus (530) Google Scholar, 3Walsh C. Nature. 2000; 406: 775-781Crossref PubMed Scopus (1199) Google Scholar, 4Leach K.L. Swaney S.M. Colca J.R. McDonald W.G. Blinn J.R. Thomasco L.M. Gadwood R.C. Shinabarger D. Xiong L. Mankin A.S. Mol. Cell. 2007; 26: 393-402Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Penicillins along with glycopeptides target peptidoglycan synthesis of the bacterial cell wall (5Wilke M.S. Lovering A.L. Strynadka N.C. Curr. Opin. Microbiol. 2005; 8: 525-533Crossref PubMed Scopus (285) Google Scholar, 6Kahne D. Leimkuhler C. Lu W. Walsh C. Chem. Rev. 2005; 105: 425-448Crossref PubMed Scopus (521) Google Scholar). Numerous small molecule nucleoside and nucleotide inhibitor analogs have been synthesized to target the essential enzymes of the human immunodeficiency virus (HIV) 2The abbreviations used are: HIV, human immunodeficiency virus; BC251, B. circulans strain 251; CGTase, cyclodextrin glucanotransferase; epPCR, error-prone PCR; pNPG7, 0.05-3 mm 4-nitrophenyl-α-d-maltoheptaoside-4-6-O-ethylidene. (7Yin P.D. Das D. Mitsuya H. CMLS Cell Mol. Life Sci. 2006; 63: 1706-1724Crossref PubMed Scopus (44) Google Scholar). The powerful mitotic antitumor inhibitor, taxol, promotes the assembly and hyperstabilization of microtubules in the treatment of breast, lung, and ovarian cancers (8Jordan M.A. Wilson L. Nat. Rev. Cancer. 2004; 4: 253-265Crossref PubMed Scopus (3600) Google Scholar, 9Gupta Jr., M.L. Bode C.J. Georg G.I. Himes R.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6394-6397Crossref PubMed Scopus (91) Google Scholar). However, resistance to all of these effective inhibitors, due to intrinsic or acquired immunity, seems to be a mere formality (10Normark B.H. Normark S. J. Intern. Med. 2002; 252: 91-106Crossref PubMed Scopus (288) Google Scholar, 11Andersson D.I. Curr. Opin. Microbiol. 2006; 9: 461-465Crossref PubMed Scopus (327) Google Scholar). The target cell may apply numerous counteractive measures to prevent the fatal actions of inhibitors. Multidrug efflux pumps are extremely effective in the removal of inhibitors from the cell in Gram-negative bacteria (12Poole K. Curr. Opin. Microbiol. 2001; 4: 500-508Crossref PubMed Scopus (227) Google Scholar). Inhibitor modification by enzymes such as aminoglycoside acetyltransferases and phosphotransferases render aminoglycoside antibiotics ineffective (2Magnet S. Blanchard J.S. Chem. Rev. 2005; 105: 477-498Crossref PubMed Scopus (530) Google Scholar). Another form of resistance involves incorporation of single or multiple mutations at the target site of the inhibitors, ultimately leading to non-adherence and lack of inhibitor potency. Mutated variants must, however, preserve cell function while acquiring resistance. Studies of clinical isolates have shown that the biological cost of native enzyme function by the acquisition of antibiotic resistance is a main determinant of both the rate and the extent of resistance development under a given antibiotic pressure (11Andersson D.I. Curr. Opin. Microbiol. 2006; 9: 461-465Crossref PubMed Scopus (327) Google Scholar). Although compensatory mutations may aid in lowering this biological cost, there is ultimately a price to be paid in the initial acquisition of this newly attained function (13Gagneux S. Long C.D. Small P.M. Van T. Schoolnik G.K. Bohannan B.J. Science. 2006; 312: 1944-1946Crossref PubMed Scopus (489) Google Scholar, 14McCallum N. Karauzum H. Getzmann R. Bischoff M. Majcherczyk P. Berger-Bachi B. Landmann R. Antimicrob. Agents Chemother. 2006; 50: 2352-2360Crossref PubMed Scopus (66) Google Scholar, 15Wichelhaus T.A. Boddinghaus B. Besier S. Schafer V. Brade V. Ludwig A. Antimicrob. Agents Chemother. 2002; 46: 3381-3385Crossref PubMed Scopus (83) Google Scholar). To investigate the delicate balance between this newly attained function and native function and stability, we have evolved a cyclodextrin glucanotransferase (CGTase) enzyme toward resistance to the small molecule inhibitor of the protein, acarbose (Fig. 1). CGTase is a well studied model enzyme for the glycoside hydrolase family 13 (GH13) (16Stam M.R. Danchin E.G. Rancurel C. Coutinho P.M. Henrissat B. Protein Eng. Des. Sel. 2006; 19: 555-562Crossref PubMed Scopus (439) Google Scholar, 17Kelly R.M. Leemhuis H. Dijkhuizen L. Biochemistry. 2007; 46: 11216-11222Crossref PubMed Scopus (51) Google Scholar, 18Janecek S. Svensson B. MacGregor E.A. Eur. J. Biochem. 2003; 270: 635-645Crossref PubMed Scopus (105) Google Scholar) catalyzing the formation of α-(1,4)-linked oligosaccharides (cyclodextrins) from starch (Fig. 1) and is strongly inhibited by the small molecule acarbose (19Leemhuis H. Dijkstra B.W. Dijkhuizen L. Eur. J. Biochem. 2003; 270: 155-162Crossref PubMed Scopus (39) Google Scholar, 20Wehmeier U.F. Piepersberg W. Appl. Microbiol. Biotechnol. 2004; 63: 613-625Crossref PubMed Scopus (167) Google Scholar). To evolve this enzyme toward acarbose insensitivity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone (ep) PCR. CGTase variants were subsequently screened for native cyclodextrin-forming activity in the presence of high acarbose concentrations. Our results demonstrate the relative ease at attaining acarbose-resistant CGTase mutants, increasing the IC50 value for acarbose up to 6,700-fold. However, detailed analysis of the insensitive variants highlights the conflicting compromise between native and newly attained enzyme function and subsequent impact on protein stability. Bacterial Strains, Plasmids, and Growth Conditions—Escherichia coli strain MC1061 was used for DNA manipulations and library screening. Plasmid-carrying strains were grown on LB medium at 37 °C in the presence of kanamycin (50 μg/ml for E. coli and 5 μg/ml for Bacillus subtilis). Bacillus circulans 251 (BC251) CGTase proteins were produced from plasmid pDP66k- using B. subtilis strain DB104A as host, as described (19Leemhuis H. Dijkstra B.W. Dijkhuizen L. Eur. J. Biochem. 2003; 270: 155-162Crossref PubMed Scopus (39) Google Scholar). Purity and molecular weight were checked by SDS-PAGE. Enzyme concentrations were determined using the Bradford reagent from Bio-Rad (München, Germany) and bovine serum albumin as standard. Saturation and Site-directed Mutagenesis—Mutants were constructed in pDP66k- as described (21Leemhuis H. Dijkstra B.W. Dijkhuizen L. FEBS Lett. 2002; 514: 189-192Crossref PubMed Scopus (49) Google Scholar) and verified by DNA sequencing (BaseClear, Leiden, the Netherlands). Construction of the single mutants A230V, K232E, F283L, I61V, D313E, and D319E was carried out using the following oligonucleotides: 5′-GC CTG CTT GAT TTA CGT TTT GC-′3 (F283L); 5′-TGG ATG CGG TG GAG CAT A-′3 (K232E); 5′-AC AAA GTC AAC GAC GG TTA C-′3 (I61V); 5′-TCC GCA GCC GAA TAC GCC CA-′3 (D313E); 5′-CGC ATG GAT GTG GTG AAG CAC ATG CCG TTC G-′3 (A230V); 5′-AT GAA CAG GTG ACG TTC ATC-′3 (D319E). The underlined regions of the oligonucleotides indicate where the nucleotide substitution was introduced. Double mutants H140Q/F283L, A230V/F283L, and A230V/H140Q were subsequently constructed using H140Q and A230V oligonucleotides with F283L and H140Q mutants as PCR template. His-140 was replaced by all 19 other amino acid residues by site-saturation mutagenesis, using the oligonucleotide: H140X, 5′-TTT GCC CCG AAC NNS ACG TC-′3. The underlined region indicates where the nucleotide substitutions were introduced. N is A+G+C+T, S is G+C, and X is any amino acid residue. Error-prone PCR Mutagenesis—The cgt gene was amplified from pDP66k- with the primers F1 (XhoI), 5′-GCG CCG GAT ACC TCG AGT TCC AAC AAG CAA AAT TTC-′3 and Rev1 (KpnI), 5′-CCA ATT CAC GTT AAT GGT ACC GGT GCC GCT GGA CGG-′3. The XhoI and KpnI restriction sites introduced (underlined) into pDP66k- resulted in V6S (N terminus) and A678G (C terminus) mutations, which had no effect on the catalytic properties of the enzyme (22Leemhuis H. Rozeboom H.J. Wilbrink M. Euverink G.-J.W. Dijkstra B.W. Dijkhuizen L. Biochemistry. 2003; 42: 7518-7526Crossref PubMed Scopus (60) Google Scholar). PCR mixtures (50 μl) contained: 1× TaqDNA polymerase buffer, 1 mm MgSO4, 0.25 mm MnCl2, 0.6 mm of each dNTP, 0.07 μm of each primer, 20 ng of pDP66k-, and 2.5 units of TaqDNA polymerase (Roche Applied Science). PCR reactions were performed for 25 cycles: 30 s 94 °C, 40 s 54 °C, and 2 min 72 °C. The PCR products were restricted with XhoI and KpnI, and the resulting fragment (2100 bp) was extracted from agarose gel (QIAquick gel extraction kit; Qiagen) and cloned in pDP66k-, replacing the wild-type cgt gene. Gene Shuffling—DNA shuffling of the single variants (A230V, F283L, K232E, H140Q) and wild-type BC251 cgt genes was carried out using an adapted version applied by Kikuchi et al. and Kaper et al. (23Kikuchi M. Ohnishi K. Harayama S. Gene (Amst.). 1999; 236: 159-167Crossref PubMed Scopus (97) Google Scholar, 24Kaper T. Brouns S.J. Geerling A.C. De Vos W.M. Van Der O.J. Biochem. J. 2002; 368: 461-470Crossref PubMed Google Scholar). Wild-type and mutant genes were amplified using the flanking primers FLKF, 5′-GGA CAA GCC TGG AAT TCA-′3, and FLKR, 5′-CCG AAG CTT GCT CAA TCA-′3. PCR products were subsequently diluted to a concentration of 84 μg/ml before being pooled. Separate overnight restriction digestions of the pooled variants by MwoI, MspI, TaqI/EcoRII, NciI/Sau3AI, and HaeIII/NciI were followed by thermal enzyme inactivation. DNA fragments were reassembled in the following PCR cycles lacking primers: 96 °C, 90 s; 35 cycles of (94 °C, 30 s; 65 °C, 90 s; 62 °C, 90 s; 59 °C, 90 s; 56 °C, 90 s; 53 °C, 90 s; 50 °C, 90 s; 47 °C, 90 s; 44 °C, 90 s; 41 °C, 90 s; 72 °C, 4 min); 72 °C, 7 min; 4 °C 10 min. One μl of this reaction was used for generation of the full-length cgt gene by PCR using the F1 and Rev1 primers (mentioned above). The resulting full-length gene products were cloned into the expression vector to obtain the second generation library. Selection of Acarbose-insensitive CGTase Variants—E. coli MC1061 cells were transformed with the epPCR library and plated on LB agar plates. Resulting colonies were transferred to 200 μl of LB medium in 96-well microtiter plates using the Q-pix (Genetix, New Milton Hamsphire, UK) and incubation overnight (750 rpm) at 37 °C. From each well, 25 μl of culture was transferred to a second 96-well plate containing 25 μl/well of bacterial protein extraction reagent (Pierce) to lyse the cells. Subsequently, 200 μl of 1% (w/v) Paselli SA2 starch with 250 μm acarbose (Serva Electrophoresis, Heidelberg, Germany) in 10 mm sodium citrate buffer (pH 6.0) was added, and the microtiter plates were incubated at 50 °C for 5 h in an oven. Under these conditions, wild-type CGTase displayed no detectable cyclization activity. The amount of β-cyclodextrin formed was measured by the addition of 10 μl of the reaction to 100 μl of phenolphthalein solution before reading absorbance at 552 nm (25Vikmon, M. (1982) Rapid and Simple Spectrophotometric Method for Determination of Microamounts of Cyclodextrins (Szejlti, J., ed) pp. 69-74, Reidel Publishing Co, Dordrecht, The NetherlandsGoogle Scholar). Enzyme Assays—All CGTase enzyme assays (initial rates) were performed in 10 mm sodium citrate buffer (pH 6.0) at 60 °C. β-Cyclodextrin-forming activity was determined by incubating 1.3-66 nm of enzyme with a 2.5% (w/v) solution of partially hydrolyzed potato starch with an average degree of polymerization of 50 (Paselli SA2; AVEBE, Foxhol, The Netherlands). The amount of β-cyclodextrin produced was quantified with phenolphthalein (25Vikmon, M. (1982) Rapid and Simple Spectrophotometric Method for Determination of Microamounts of Cyclodextrins (Szejlti, J., ed) pp. 69-74, Reidel Publishing Co, Dordrecht, The NetherlandsGoogle Scholar). Inhibition by acarbose (IC50) was determined directly from the cyclization assay in the presence of 0.2-30,000 μm acarbose. The data were fitted to a four-parameter non-linear regression function using SigmaPlot 10 software (Systat). E=max+((min−max))/(1+C/IC50)k))+max,(Eq. 1) where min = minimum response, max = maximum response, C = acarbose concentration, IC50 = acarbose concentration causing half-maximum β-cyclization activity, and k = curve slope. Disproportionation activity was measured as described (19Leemhuis H. Dijkstra B.W. Dijkhuizen L. Eur. J. Biochem. 2003; 270: 155-162Crossref PubMed Scopus (39) Google Scholar), using 0.06-0.6 nm enzyme, 0.05-3 mm 4-nitrophenyl-α-d-maltoheptaoside-4-6-O-ethylidene (pNPG7; Megazyme, Wicklow, Ireland) as donor substrate, and 0.05-3 mm maltose as acceptor substrate. The 4-hydroxyl of the donor substrate pNPG7 is blocked, thereby preventing the donor substrate from being used as acceptor substrate. Fitness of epPCR Library—Competent E. coli MC1061 cells were transformed with the epPCR plasmid library, and the entire transformation (∼2 million transformants) was used to inoculate 2 liters of LB medium containing 50 μg/ml kanamycin. Following overnight growth at 37 °C, cells were collected by centrifugation. Cells were broken by French press (10,000 p.s.i), and cell debris was removed by ultracentrifugation at 4 °C for 30 min at 15,000 × g. CGTase proteins were subsequently purified as described previously (22Leemhuis H. Rozeboom H.J. Wilbrink M. Euverink G.-J.W. Dijkstra B.W. Dijkhuizen L. Biochemistry. 2003; 42: 7518-7526Crossref PubMed Scopus (60) Google Scholar). High Pressure Liquid Chromatography Analysis—Formation of cyclodextrins from starch (10% (w/v) Paselli SA2 in 10 mm sodium citrate buffer, pH 6.0) was analyzed by incubating the starch for 54 h with 13 nm wild-type and 65 nm mutant proteins (A230V, H140Q, K232E, F283L, and A230V/H140Q). Samples were taken at taken at regular time intervals and subsequently boiled for 30 min for enzyme inactivation. Products formed were analyzed on a homemade Benson BC calcium column (300 × 7.8 mm ID) at 90 °C connected to a refractive index detector. A mobile phase of 100 ppm Ca2+-EDTA in demineralized water at a flow rate of 0.2 ml/min was used. Differential Scanning Calorimetry—Thermal unfolding of (mutant) CGTases was measured using a MicroCal VP-DSC microcalorimeter (MicroCal Inc., Northhampton, MA) with a cell volume of 0.52 ml. Experiments were performed at a scan rate of 1 °C/min at a constant pressure of 2.75 bars. Samples were degassed prior to scan. The enzyme concentration used was 6.9 μm in 10 mm sodium acetate buffer, pH 5.5 (26Leemhuis H. Rozeboom H.J. Dijkstra B.W. Dijkhuizen L. Proteins. 2004; 54: 128-134Crossref PubMed Scopus (38) Google Scholar). Generation of Acarbose-insensitive Mutant CGTase Proteins—Random genetic diversity was created by error-prone PCR amplification of the cgt gene in the presence of 0.25 mm MnCl2. Under these conditions, 90% of the E. coli transformants retained starch-degrading activity as detected by the formation of halos surrounding the colonies on starch/agar plates. The fitness of the epPCR library was 60% regarding the initial β-cyclization activity, and the IC50 value for acarbose was 1.2 μm, nearly identical to that of the wild-type enzyme (Table 1). Twelve thousand variants of the library were screened for decreased inhibition by acarbose. Fifty-six variants retained β-cyclization activity in the presence of 250 μm of the acarbose inhibitor. Sequencing of 13 variants revealed the presence of A230V (9×), K232E (2×), and F283L (2×) mutations. Also, I61V, D313E, and D319E mutations were found, but only in combination with those mentioned. Construction and characterization of the purified proteins of the single mutations I61V, D313E, D319E, A230V, K232E, and F283L demonstrated that the A230V, K232E, and F283L mutations were responsible for CGTase insensitivity to acarbose inhibition (Table 1). Surprisingly, no His-140 mutant was identified from the screening as this histidine residue has been shown to be important for the strong inhibition by acarbose in Bacillus sp. 1011 and Thermoanaerobacterium thermosulfurigenes EM1 CGTases (27Nakamura A. Haga K. Yamane K. Biochemistry. 1993; 32: 6624-6631Crossref PubMed Scopus (98) Google Scholar, 28Leemhuis H. Wehmeier U.F. Dijkhuizen L. Biochemistry. 2004; 43: 13204-13213Crossref PubMed Scopus (25) Google Scholar). His-140 was therefore targeted by saturation mutagenesis. Screening of the 288 clones from the H140X library yielded six variants that formed β-cyclodextrins in the presence of 250 μm acarbose. Three of the clones were randomly picked and sequenced, revealing the His to Gln substitution in each case.TABLE 1Cyclization rates from starch and IC50 values for acarbose by wild-type and acarbose insensitive CGTase mutants from Bacillus circulans 251Enzymeβ-Cyclization kcatIC50-Fold increasea-Fold increase indicates the factor of improvement of variants compared to wild-type based on IC50 valuess–1μmWild-type329 ± 31.11EpPCR librarybMixture of mutant CGTases from the epPCR library195 ± 51.21.1A230V9 ± 0.17,3706,700H140Q79 ± 31,8071,642F283L35 ± 0.54,1973,815K232E26 ± 13,9303,572A230V/H140Q48 ± 21,8451,677a -Fold increase indicates the factor of improvement of variants compared to wild-type based on IC50 valuesb Mixture of mutant CGTases from the epPCR library Open table in a new tab Shuffling and Construction of Double Mutant CGTase Variants—In an effort to further decrease the inhibitory effects by acarbose, while retaining or increasing native β-cyclization activities, DNA shuffling of the selected variants from the error-prone library was carried out. Over 8,000 clones were screened at an increased concentration of 500 μm acarbose; however, no improved variants were found. To investigate whether combinations of mutations selected in the first round had additive effects for CGTase insensitivity toward acarbose, the double mutants A230V/F283L, A230V/H140Q, and F283L/H140Q were constructed. Only the A230V/H140Q mutant remained functional in β-cyclization production, with lower acarbose resistance levels when compared with the single mutants generated by epPCR (Table 1). This may explain the lack of identification of better performing mutants from shuffling. IC50 Values of Wild-type and Mutant CGTases—Measurement of β-cyclization activity in the presence of varying concentrations of acarbose revealed that mutations at the acceptor subsites of CGTase had a profound effect on inhibitor insensitivity, raising IC50 values by 3,500-6,700-fold when compared with wild type (Table 1). Mutation H140Q, located at the -1 donor subsite, had the least effect on inhibitor resistance, although still increasing the IC50 value over 1,600-fold when compared with wild type (Table 1). Catalytic Properties of Wild-type and Mutant CGTases—The β-cyclization rates of the selected mutations were compromised (Table 1). The F283L, K232E, and A230V/H140Q variants showed comparable decreases of ∼10-fold in the initial β-cyclization rates when compared with wild type, whereas the A230V mutation was most severely affected with an almost 40-fold reduction in activity. Product analysis also revealed a 3.4-fold reduction in cyclodextrin production for the A230V mutant, with a large increase in short linear saccharide formation when compared with wild type (not shown). All other mutants formed approximately the same amount of β- and γ-cyclodextrins as wild type, with lowered production of α-cyclodextrin (Fig. 2). How the mutations at the subsites -1/+1/+2 alter cyclodextrin product specificity is not yet clearly understood (29Leemhuis H. Dijkhuizen L. Biocatal. Biotransform. 2003; 21: 261-270Crossref Scopus (11) Google Scholar). Measurements of the catalytic efficiency (kcat/Km) for processing of the maltoheptaoside substrate pNPG7 in the disproportionation reaction with maltose as acceptor substrate revealed a 5-fold (F283L) to 18-fold (H140Q and K232E) reduction (Table 2). Surprisingly, the catalytic efficiency of the A230V/H140Q combination is much higher than that of its single mutant counterparts. The Km values for the acceptor substrate maltose were not significantly altered by most mutations, with only the H140Q mutant displaying a 2-fold decrease when compared with wild type (Table 2).TABLE 2Kinetic parameters of the disproportionation reaction of wild-type and mutant B. circulans 251 CGTases with the blocked pNPG7 and maltose substratesEnzymekcatKm, pNPG7Km, maltosekcat/Km, pNPG7s–1mmmms–1/mmWild type490 ± 100.09 ± 0.011.2 ± 0.175,444A230VaDue to elevated hydrolytic rates of A230V, only hydrolysis of pNPG7 could be measured. The presence of maltose had no effect on the rate of pNPG7 degradation45 ± 20.30 ± 0.04150H140Q77 ± 20.26 ± 0.010.48 ± 0.03296F283L136 ± 50.11 ± 0.011.4 ± 0.111,236K232E84 ± 10.27 ± 0.011.2 ± 0.17311A230V/H140Q144 ± 50.09 ± 0.011.9 ± 0.071,600a Due to elevated hydrolytic rates of A230V, only hydrolysis of pNPG7 could be measured. The presence of maltose had no effect on the rate of pNPG7 degradation Open table in a new tab Stability of Wild-type and Mutant CGTases—To investigate whether the mutations selected for their capacity to minimize acarbose inhibition affected the stability of the enzyme, the CGTase variants were denaturated by heat using differential scanning calorimetry. Both the wild-type and the mutant proteins displayed irreversible thermal unfolding patterns. All mutants were significantly affected in stability, lowering the apparent melting temperature between 7 and 11 °C (Fig. 3). Mechanism and Effects of Acquired Inhibitor Resistance—As a general trend, most resistance-conferring mutations are located throughout the active site due to the substrate mimicking nature of small molecule inhibitor (Table 3). As active sites have primarily evolved for reaction specificity and rate enhancement, introduction of such mutations is expected to have a negative effect on the catalytic efficiency of the enzyme. A trade-off between enzyme function and inhibitor resistance is indeed observed for enzymes with resistance-conferring mutations (14McCallum N. Karauzum H. Getzmann R. Bischoff M. Majcherczyk P. Berger-Bachi B. Landmann R. Antimicrob. Agents Chemother. 2006; 50: 2352-2360Crossref PubMed Scopus (66) Google Scholar, 15Wichelhaus T.A. Boddinghaus B. Besier S. Schafer V. Brade V. Ludwig A. Antimicrob. Agents Chemother. 2002; 46: 3381-3385Crossref PubMed Scopus (83) Google Scholar, 30Marciano D.C. Karkouti O.Y. Palzkill T. Genetics. 2007; 176: 2381-2392Crossref PubMed Scopus (37) Google Scholar, 31Fisher T.S. Joshi P. Prasad V.R. J. Virol. 2002; 76: 4068-4072Crossref PubMed Scopus (39) Google Scholar, 32Cong M.E. Heneine W. Garcia-Lerma J.G. J. Virol. 2007; 81: 3037-3041Crossref PubMed Scopus (100) Google Scholar, 33Bjorkholm B. Sjolund M. Falk P.G. Berg O.G. Engstrand L. Andersson D.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14607-14612Crossref PubMed Scopus (298) Google Scholar, 34Fernandez A. Tawfik D.S. Berkhout B. Sanders R. Kloczkowski A. Sen T. Jernigan B. J. Biomol. Struct. Dyn. 2005; 22: 615-624Crossref PubMed Scopus (14) Google Scholar, 35Aharoni A. Gaidukov L. Khersonsky O. McQ Gould S. Roodveldt C. Tawfik D.S. Nat. Genet. 2005; 37: 73-76Crossref PubMed Scopus (655) Google Scholar) (Table 3). Directed evolution of our model enzyme, BC251 CGTase, also resulted in most effective resistance-conferring mutations (A230V, K232E and F283L) clustered in and around the active site (Fig. 4). All three residues (Ala-230, Lys-232, and Phe-283) are conserved among CGTases (supplemental Table S1), indicating their important role in enzyme catalysis and reaction specificity. Replacement of such essential residues yielded variants with severely compromised catalytic function (Tables 1 and 2).TABLE 3Effects of evolved variants toward small molecule inhibitor insensitivity, on native enzyme function and stability · indicates active site.Enzyme (Inhibitor)Mutations (location)-Fold reductiona-Fold reduction indicates the factor of reduced inhibitor sensitivity of variants compared to wild-type based on IC50 or Ki valuesCost to nativebComparison between variants and wild-type based on kcat/Km for enzymatic activities and indicated thermostability testsReferenceActivityStabilityThymidylate synthase (5-fluorodeoxyuridine)T51S ·110.5653Kawate H. Landis D.M. Loeb L.A. J. Biol. Chem. 2002; 277: 36304-36311Abstract Full Text Full Text PDF PubMed Scopus (45) Google ScholarHIV reverse transcriptase (DNA aptamer, RT1t49)N255D/N265D ·1500.4331Fisher T.S. Joshi P. Prasad V.R. J. Virol. 2002; 76: 4068-4072Crossref PubMed Scopus (39) Google ScholarGlutamate-1-semialdehyde aminotransferase (4-amino-5-fluoro pentanoic acid)S163T ·>16.60.0854Gough K. Allison G. Rogers L. Smith A. Eur. J. Phycol. 2002; 37: 419-428Crossref Scopus (3) Google ScholarHepatitis C Virus Serine Protease (Protease Inhibitor BILN 2061)D168A ·>2000.16755Lin C. Lin K. Luong Y.P. Rao B.G. Wei Y.Y. Brennan D.L. Fulghum J.R. Hsiao H.M. Ma S. Maxwell J.P. Cottrell K.M. Perni R.B. Gates C.A. Kwong A.D. J. Biol. Chem. 2004; 279: 17508-17514Abstract Full Text Full Text PDF PubMed Scopus (271) Google ScholarTaq DNA polymerase (Heparin)K225E/E388VcContains additional mutations D578G/N583S/M747R/K540R ·2600.720.2dThermostability-activity half-life (t½) at 95 °C56Ghadessy F.J. Ong J.L. Holliger P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4552-4557Crossref PubMed Scopus (309) Google ScholarHIV-1 protease (p2-NC analog)G48V ·270.550.35eRatio of the urea concentrations at half maximal activity57Mahalingam B. Louis J.M. Reed C.C. Adomat J.M. Krouse J. Wang Y.F. Harrison R.W. Weber I.T. Eur. J. Biochem. 1999; 263: 238-245Crossref PubMed Scopus (115) Google ScholarIsoleucyl-tRNA synthetase (Mupirocin)H594Y ·>43f-Fold increase in MIC (μg ml–1) of mupirocin for Salmonella typhimurium growth0.4258Paulander W. Maisnier-Patin S. Andersson D.I. Mol. Microbiol. 2007; 64: 1038-1048Crossref PubMed Scopus (51) Google ScholarHIV-1 protease (Indinavir)I50V ·500.180.6eRatio of the urea concentrations at half maximal activity59Liu F. Boross P.I. Wang Y.F. Tozser J. Louis J.M. Harrison R.W. Weber I.T. J. Mol. Biol. 2005; 354: 789-800Crossref PubMed Scopus (61) Google ScholarROB-1 β-lactamase (Clavulanate)S130G ·1660.1560Galan J.C. Morosini M.I. Baquero M.R. Reig M. Baquero F. Antimicrob. Agents Chemother. 2003; 47: 2551-2557Cros