Directed Evolution of D-Sialic Acid Aldolase to L-3-Deoxy-Manno-2-Octulosonic Acid (L-KDO) Aldolase

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Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase

Che-Chang Hsu, Zhangyong Hong, Masaru Wada, Dirk Franke, and Chi-Huey Wong*

Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Chi-Huey Wong, May 14, 2005

An efficient L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase was created by directed evolution from the Escherichia coli D-Neu5Ac (N-acetylneuraminic acid, D-sialic acid) aldolase. Five rounds of error-prone PCR and iterative screening were performed with sampling of 103 colonies per round. The specificity constant (kcat͞Km) of the unnatural sugar L-KDO is improved to a level equivalent to the wild-type D-sialic acid aldolase for its natural substrate, D-Neu5Ac. The final evolved enzyme exhibits a >1,000- fold improved ratio of the specificity constant [kcat͞Km (L-KDO)]͞ [kcat͞Km (D-sialic acid)]. The protein sequence of the evolved aldolase showed eight amino acid changes from the native enzyme, with all of the observed changes occurring outside of the active site. Our effort demonstrates that an enzyme can be rapidly altered to accept enantiomeric substrates with screening of a small population of colonies iteratively toward the target substrate with improved catalytic efficiency. This work provides a method for the synthesis of enantiomeric sugars and for the study of enantiomeric catalysis affected by remote mutations.

inversion of enanselectivity ͉ enzyme engineering ͉ L-sugar synthesis Fig. 1. In vitro evolution of D-peptide binders that target cell-surface sugars. arbohydrates constitute unique cellular structures that play Ca vital role in various molecular recognition processes. representing the most common structural motif for aldolases (8). Interference of this recognition process presents a great poten- Despite a few efforts to understand the catalytic mechanism of tial in biomedical and pharmaceutical applications. Thus, devel- sialic acid aldolase (7, 9), rational design of the enzyme for a oping methods of interference-targeting cell-surface carbohy- reversal of enantioselectivity remains a tremendous challenge drates may offer therapeutic candidates for treatment of (Fig. 2). Using the approach of directed evolution by means of inflammation, cancer metastasis, and bacterial or viral infection iterative in vitro screening, it has been demonstrated that enzyme (1). Previously, we developed a strategy in which enantiomers of activity can be rapidly tuned to alter substrate specificity or to natural peptides are identified to target cell-surface sugars invert enantioselectivity without prior structure-function knowl- through in vitro phage selection (2) (Fig. 1). These D-peptide edge (10–15). Our efforts in this regard have improved D-2- binders of cell-surface carbohydrates provide a valuable tool for keto-3-deoxy-6-phosphogluconate aldolase toward accepting the the study of protein and carbohydrate interactions. With this phosphate-free L-enantiomeric substrate, L-glyceraldehyde, in- approach, we have identified a dodecapeptide binding to sialic stead of the natural D-glyceraldehyde 3-phosphate with retention acid with a dissociation constant (Kd)ofϷ0.5 mM. Another of facial selectivity in the step of C-C bond formation. We have target of interest is D-3-deoxy-manno-2-octulosonic acid (D- also altered the enantioselectivity of sialic acid aldolase for KDO), which is an essential component of the outer-cell mem- N-acetylneuraminic acid and KDO (11). brane lipopolysaccharide of Gram-negative bacteria. These D- peptide binders of D-KDO may offer previously unrecognized Materials and Methods antibacterial agents. A prerequisite to this approach is an General. Standard molecular biology techniques and the screen- efficient synthesis of the enantiomeric L-sugars of the natural ing assays were followed with moderate modifications (11, 16). D-configured carbohydrates. The current preparative use of the Restriction endonucleases and T4 DNA ligase were purchased wild-type N-acetylneuraminic acid aldolase (sialic acid aldolase, from New England Biolabs. Taq and Pfu polymerases were EC 4.1.3.3) for the synthesis of L-sialic acid and other high- purchased from Stratagene. UV kinetic assays were performed carbon L-sugars is hampered by its low turnover rate; therefore, on a Beckman DU-650 spectrophotometer. Fluorescence kinetic improving sialic acid aldolase toward accepting the unnatural measurements were performed on an F2000 fluorescence spec- L-enantiomeric substrate is needed to facilitate its application in trophotometer (Hitachi, Tokyo). NMR spectra (1 H, 400 MHz) synthetic chemistry. were recorded on an AMX-400 spectrometer (Bruker, Billerica, N-acetyl-D-neuraminic acid aldolase (sialic acid aldolase) MA). Mass spectra were recorded on a Hitachi M-8000 liquid catalyses the reversible aldol reaction of N-acetyl-D-man- chromatography-mass spectrometer under sonic or electrospray nosamine and pyruvate to produce N-acetyl-D-neuraminic acid conditions. DNA sequencing was performed on an Applied (D-sialic acid) (Fig. 2). D-sialic acid is an essential cell-surface Biosystems 377 DNA sequencer automated sequencer, using residue associated with inflammatory diseases, cancer metastasis, and influenza virus infection (3, 4). A number of sialic acid analogs have been synthesized for their numerous biological and Abbreviation: KDO, 3-deoxy-manno-2-octulosonic acid. pharmaceutical applications (5, 6). The structure of the sialic *To whom correspondence should be addressed. E-mail: [email protected]. acid aldolase is composed of eight repeating ␣͞␤ barrels, (7) © 2005 by The National Academy of Sciences of the USA

9122–9126 ͉ PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504033102 Downloaded by guest on September 26, 2021 Fig. 2. Directed evolution of D-sialic acid aldolase to L-KDO aldolase. Fig. 3. D- and L-KDO cleavage speciﬁcity constant of sialic acid aldolase variants through the progress of directed evolution. thermal cycle sequencing conditions with fluorescently labeled terminators. Curve fitting was done by the nonlinear least- with mild shaking. Mutants that had the highest activity from squares method using GRAFIT (Erithacus Software, Surrey, each selected clone were replicated into eight individual plates U.K.). Samples on 96-well plates were analyzed with a fluores- and rescreened to help eliminate false positives. The active cence spectrophotometer (Fusion Universal Microplate Ana- mutants thus selected were subsequently cultured and overex- lyzer, Packard) for microtiter plate assays. pressed in 1-liter prep and purified with a Ni2ϩ-nitrilotriacetic acid agarose column. The retro-aldol enzymatic assays of mu- Mutant Library Preparation and Assay. Sialic acid aldolase (nanA tants with various substrates were coupled to NADH depletion gene) was amplified from genomic DNA of Escherichia coli and as described above. The enzymatic aldol condensation activity cloned into vector pTrcHisB (Invitrogen). Mutagenic PCR was was determined by the rate of pyruvate depletion as reported in carried out under standard error-prone conditions, and the ref. 10. constructs harboring a mutant library of N-acetylneuraminic acid aldolase were transformed into JM109-competent cells by Molecular Modeling and Substrate Docking. Based on protein se- electroporation. The transformed culture was plated on LB agar quence homology and structure similarities, the crystal structure plates containing 100 ␮g͞ml ampicillin and incubated overnight of E. coli sialic acid aldolase was superimposed on influenza A at 37°C. Individual colonies were selected and inoculated into N9 neuraminidase with an analog of sialic acid bound to it (17). CHEMISTRY 96-well plates containing 0.5 ml of LB and 50 ␮g͞ml ampicillin With the structural alignment of these two enzymes, the sialic and sealed with gas-permeable membranes (Breathe Easy, Di- acid analog, 4-deoxy-N-acetyl neuraminic acid, initially bound to versified Biotech). They were grown overnight at 37°C and influenza A N9 neuraminidase, is now also docked in the active shaken at 250 rpm to reach uniform cell density. Then, 10 ␮lof site of the E. coli sialic acid aldolase. Our structural model the overnight precultures were inoculated into expression cul- reveals the active site of wild-type sialic acid aldolase bound to tures in 96-well, 2-ml-deep well plates containing 0.5 ml of LB 4-deoxy-N-acetyl neuraminic acid with influenza A N9 neur- and 50 ␮g͞ml ampicillin. The preculture plates were stored at aminidase concealed in the background (Fig. 5). Protein struc- 4°C for rescreening of active clones. The expression cultures tural alignment was produced with SWISS-PDBVIEWER and ren- were grown to a cell density of OD600 ϭ 0.5, and 0.5 ml of LB dered with PYMOL. containing 50 ␮g͞ml ampicillin and 1 mM isopropyl ␤-D- thiogalactoside was added to the wells. After6hofgrowth,the Preparation of Substrates. N-acetyl-D-neuraminic acid was a gift plates were centrifuged at 10,000 ϫ g at 4°C for 1 h, and the from Taiyo Kagaku (Yokkaichi, Japan). N-acetyl-L-neuraminic supernatant was carefully decanted. Each cell pellet was resus- acid was synthesized as previously described with the exception pended in 0.5 ml of 50 mM potassium phosphate buffer (pH 7.5) that the starting materials were replaced by its L-enantiomeric containing 0.5 mg͞ml lysozyme. The plates were rapidly frozen counterparts (18). N-acetyl-D-mannosamine, D-mannosamine, in liquid nitrogen, thawed at 4°C in a sonicator bath, and D-mannose, L-mannose, D-arabinose, L-arabinose, D-gulose, L- incubated at 65°C for 20 min. Cell debris was collected by gulose, D-talose, D-xylose, D-glucose, and N-acetyl-D- centrifugation at 10,000 ϫ g at 4°C for 1 h, and 100 ␮lofthe glucoseamine were purchased from Sigma-Aldrich. D-KDO and supernatant from each well was transferred into black polysty- L-KDO were first enzymatically synthesized with the wild-type rene 96-well plates. Upon incubation at 25°C for 10 min, 40 ␮l sialic acid aldolase and then later replaced by mutants with of an assay solution containing 150 mM potassium phosphate, improved activity toward both enantiomers of KDO. KDO was NADH, and L-lactic dehydrogenase at pH 7.5 was dispensed into subsequently purified by AG-1 X-8 (formate) anion-exchange each well. The starting concentration of NADH in each well was chromotography resin (Bio-Rad) and recrystallized with ethanol 360 mM, and the amount of lactic dehydrogenase was 0.1 unit. and water to remove pyruvate contamination. 3-bromo-3-deoxy- Baseline drift was monitored for 2 min. After leveling of the D-mannose and 3-bromo-3-deoxy-D-glucose were synthesized as baseline to an insignificant drift, 10 ␮lofD-KDO or L-KDO described in ref. 9. (final concentration of 13 mM for the first two generations of mutants) or N-acetyl-L-neuraminic acid (30 mM for the third- Results and Discussion generation mutants and 10 mM for the fourth-generation mu- To invert the enantioselectivity of the natural substrate of sialic tants) in 50 mM potassium phosphate (pH 7.5) was added to acid aldolase, we had initially focused on L-sialic acid, a substrate each well. The fluorescence at an excitation of 340 nm and an with a very slow turnover rate. The initial attempt was to screen absorbance of 450 nm was monitored continuously for 25 min for mutant enzymes to directly accept L-sialic acid. However, a

Hsu et al. PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 ͉ 9123 Downloaded by guest on September 26, 2021 Table 1. Kinetics parameters of wild-type and evolved sialic acid aldolase

[kcat͞Km (L-KDO)]͞ Ϫ1 Ϫ1 Ϫ1 Enzyme Substrate Km,mM kcat,s kcat͞Km,mM ⅐s [kcat͞Km (D-sialic acid)]

Wild type D-sialic acid 2.6 (0.2) 10.5 (1.8) 4.04 L-sialic acid 4.4 (3.2) 0.0002 (.0001) 0.00005 D-KDO 13.8 (1.1) 1.2 (0.15) 0.087 L-KDO 7.5 (1.2) 1.1 (0.2) 0.15 0.036 N1G8 D-sialic acid 2.1 (0.27) 10.3 (2.6) 4.98 L-sialic acid N.D N.D. N.D. D-KDO 12.3 (1.7) 1.5 (0.18) 0.12 L-KDO 14.1 (1.25) 1.1 (0.12) 0.08 0.016 N2C5 D-sialic acid 3.9 (0.45) 9.7 (0.82) 2.49 L-sialic acid 369.4 (98.3) 0.0045 (0.0031) 0.00001 D-KDO 14.5 (0.9) 2.8 (0.1) 0.19 L-KDO 9.2 (0.58) 1.9 (0.21) 0.21 0.1 N3A4 D-sialic acid 12.1 (0.82) 2.2 (0.38) 0.18 L-sialic acid 127 (17) 0.0068 (0.0021) 0.0001 D-KDO 3.5 (0.64) 3.2 (0.22) 0.91 L-KDO 2.7 (0.52) 2.5 (0.31) 0.92 5.0 N4E6 D-sialic acid 10.1 (0.28) 1.7 (0.96) 0.17 L-sialic acid 36.2 (4.5) 0.02 (0.01) 0.001 D-KDO 6.3 (1.2) 5.1 (1.6) 0.81 L-KDO 2.4 (0.22) 4.2 (0.48) 1.75 10.4 N5B2 D-sialic acid 15.3 (1.59) 1.5 (0.20) 0.10 L-sialic acid 33.5 (0.38) 0.02 (0.01) 0.001 D-KDO 11.4 (0.28) 8.9 (1.3) 0.78 L-KDO 2.8 (0.52) 10.4 (1.9) 3.71 37.9

All kinetics measurements were collected in 50 mM potassium phosphate buffer, pH 7.5͞5 mM 2-mercaptoethanol at 25°C. kcat and Km were determined by nonlinear least-squares ﬁt of the Michaelis–Menten plots using GRAFIT software. Numbers in parentheses are the standard errors of curve ﬁtting. N.D., not detectable.

slow turnover rate hindered the use of wild-type sialic acid the most active mutant containing two mutations, Y98H and F115L aldolase as the starting point for directed evolution (Table 1). In (Fig. 3). The most active mutant from the second round of addition to the slow turnover rate, L-sialic acid is a rare sugar that screening toward accepting L-KDO accumulated another mutation, is difficult to synthesize. V251I. The drawback of the epPCR mutagenic method is the biased Hence, we tried to coax the enzyme to accept L-sialic acid by mutation of an amino acid residue to about six possible variations expanding the scope of its substrate specificity with an analogue, with similar physicochemical properties, which is attributed to the L-KDO, which is easier to synthesize (11). Although enzyme constraint of a single-base mutation on a given amino acid residue. adaptation for substrate analogues is unpredictable (19), it has By exploring the full extent of amino acid combinations with these been demonstrated that activities of some substrate analogues do putative ‘‘hot spots,’’ enantioselectivity had been further improved increase throughout the course of directed evolution (10, 14, 20). (23). Thus, we used DNA shuffling (24) and saturation mutagenesis The wild-type sialic acid aldolase exhibits a low level of activity on these mutations to probe for the most beneficial combination of amino acids to further optimize mutant activity. Yet, none of the toward D- and L-KDO (5, 21). By alternating D- and L-KDO as variants from either library contained any significant improvement, a starting substrate in screening, we have selected several perhaps indicating that the synergistic effect of the original muta- mutants with improvements in KDO catalysis (11). Among the tions might have been close to the local maxima of the fitness mutants tested for L-sialic acid catalysis, a variant of sialic acid landscape. aldolase mutant (N2C5) exhibited an improved turnover rate for L-sialic acid (Table 1), which is primarily due to mutations in Y98H, F115L, and V251I. We then further evolved it toward accepting L-sialic acid. A screening methodology similar to our previous approaches was used for this study (10, 11). The detection couples aldol cleavage reaction with the reduction of pyruvate to lactate and a concomitant oxidation of NADH to NADϩ in the presence of lactic dehydrogenase. Assay samples containing L-sialic acid, the crude cell lysate mutant enzyme, and the coupled-enzyme detection system were prepared on 96-well plates. The fluorescence of NADH in each well was monitored (with excitation at 340 nm and emission at 450 nm). To explore the entire protein sequence space, error-prone PCR (epPCR) was used as the initial method of mutagenesis (22). With a controlled mutation rate of one to five bases per gene that yielded one to five amino acid changes for the protein, each library was subjected to screening of a small population of 103. The first- generation mutant screened toward accepting D-KDO resulted with Fig. 4. Sialic acid aldolase variants for the use of preparative synthesis.

9124 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504033102 Hsu et al. Downloaded by guest on September 26, 2021 Fig. 5. Active site-docked 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid with amino acid mutations in mutant aldolase variants N2C5 (Left) and N5B2 (Right).

Mutant N2C5 has a 27-fold kcat improvement but a substantial turnover rate would trap the substrate in the enzyme along with the decrease in binding affinity for L-sialic acid, which led to a 50% crystal structure. Without any success, we constructed a structural overall decrease in the specificity constant (kcat͞Km) (Table 1). model based on protein sequence homology and structural simi- Because our screening method employs a continuous kinetic assay larity of E. coli sialic acid aldolase to influenza A N9 neuraminidase, that detects the linear progression of the enzyme reaction, we used with an analog of sialic acid bound to it (17). After a structural a second-round mutant, N2C5, which has an improved kcat, instead alignment of these two enzymes along with the analog bound to of the wild-type enzyme with a smaller Km, as our starting template influenza A N9 neuraminidase, we were able to analyze the active to evolve toward L-sialic acid. Screening with low initial activity was site of E. coli sialic acid aldolase, which is now also docked with the facilitated by the presence of a sufficient amount of substrate and sialic acid analog, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid enzyme concentration: L-sialic acid concentration is set relatively (Fig. 5). Similar to our previous studies in directed evolution, the high at 30 mM. The initial rounds of screening with L-sialic acid accrued mutations do not provide definitive information toward contained predominantly false positives, a problem exacerbated by mechanistic understanding in the improvement of L-sugar catalysis. a low specificity constant and a limited amount of the target Without direct interaction with the substrate, all of the mutations substrate. Thus, improvements toward L-sialic acid in early rounds identified are present on the outside of the active site (Fig. 5). They of screening borderline the threshold of detection as defined by the also do not pertain to any topological preferences. Presumably, the ϭ ͞ ϫ coefficient of variance (CV [standard deviation mean] 100%), enzyme activity is enhanced by a synergistic influence of mutations which can fluctuate between 15% and 30%. Because the precise

at remote sites that mediated subtle changes at the active site, a CHEMISTRY assessment of enzyme activity can only be accurately verified after common observation in numerous directed evolution experiments purification of each individual mutant, false positives are often regarding activity and stability (25–27). detected at later stages of the entire process. This problem was The reverse aldol reaction between pyruvate and sugar sub- somewhat alleviated by rescreening of each initial positive clone strates has been extensively characterized with wild-type sialic with eight duplications on a 96-well plate. Selection from mutants acid aldolase (9, 28). The enzyme is selective for sugar substrates with the highest turnover rate identified N3A4, which contained an with the D-configuration; however, it also accepts several L- additional mutation of V265I and exhibited improvements in k as cat sugars at low reaction rates. To probe changes in aldol conden- well as K . In the fourth round of screening, we decreased the m sation of mutants for synthetic applications, we examined the concentration of the substrate in an attempt to further improve K m rate of addition for a few reactions (Table 2). Compared with the and were able to reduce the monitor time to select mutants with a wild-type enzyme, N4E6 and N5B2 mutants displayed a 10-fold higher turnover rate. With progressive improvements, the fourth- round screening selected a mutant (N4E6) with additional muta- improvement in the rate of addition of N-acetyl-L-mannosamine tions of Y281C and N153Y and a 126-fold improvement in k over to pyruvate. N-acetyl-L-mannosamine has been reported as a cat very weak substrate for the wild-type E. coli sialic acid aldolase. the wild-type but a modest 18-fold improvement in kcat͞Km due to We noticed an improvement in the rate of addition for D- and the relatively high Km. Interestingly, the Km has decreased 10-fold from the second-round mutant N2C5. With mutant N4E6, we have L-arabinose to pyruvate with mutant N2C5 and mutant N3A4, but a subsequent reduction was observed with mutant N4E6, and also observed a 4-fold improvement in the kcat of L-KDO cleavage then improvement was once again observed with mutant N5B2. activity, which contributed to a Ͼ10-fold improvement in kcat͞Km. With a further interest to improve upon L-sugars catalysis, we again These progressive improvements are perhaps due to substrate improved its activity toward L-KDO cleavage. Improvement in selection; mutants N2C5 and N5B2 were screened with L-KDO, L L-KDO catalysis is reflected by its Ͼ10-fold increase in kcat and a and N4E6 has been optimized toward -sialic acid. Interestingly, Ͼ30-fold increase in specificity constants with the fifth-round the addition reaction of D-gulose and pyruvate has improved mutant N5B2, with more mutations in E60A and D150G, which has concurrently with improvement of N-acetyl-L-mannosamine and an overall 1,000-fold improved ratio of the specificity constant pyruvate activity. With structural comparison of D-mannose and [kcat͞Km (L-KDO)]͞[kcat͞Km (D-sialic acid)]. D-gulose, we noticed that D-gulose is almost a mirror image of To analyze the amino acid changes in mutant variants, we have D-mannose, with the only difference being in the stereochemistry mapped the mutations on the crystal structure of E. coli sialic acid at C5. Along with L-mannose, these are the two analogues that aldolase. However, the only crystal structures of E. coli sialic acid most resemble the structure of N-acetyl-L-mannosamine, which aldolase to date have only pyruvate analogues bound to the active is the educt of the natural enantiomeric substrate, L-sialic acid. site, thus rendering insufficient information on the substrate and Clearly, the mutant activity of these two substrates closely active site interaction of the nine-carbon sialic acid or the eight- mimics the improvement in the activity of N-acetyl-L- carbon KDO. Therefore, we tried to cocrystallize either L-KDO or mannosamine and pyruvate, as well as L-sialic acid cleavage L-sialic acid with E. coli sialic acid aldolase, hoping that the slow activity (Table 1).

Hsu et al. PNAS ͉ June 28, 2005 ͉ vol. 102 ͉ no. 26 ͉ 9125 Downloaded by guest on September 26, 2021 Table 2. Relative rates of substrates for sialic acid aldolase variants Acceptor substrate Wild type N2C5 N3A4 N4E6 N5B2

N-acetyl-D-mannosamine 100 100 100 100 100 N-acetyl-L-mannosamine 0.2 0.5 1.0 2.0 2.0 D-mannosamine 80 90 165 190 170 D-mannose 80 110 95 95 95 L-mannose 30 15 70 80 75 D-arabinose 20 35 70 40 80 L-arabinose 10 40 65 40 85 D-gulose 10 N.D. 110 90 70 L-gulose 70 35 15 15 15 D-talose 60 50 105 80 70 D-glucose 40 40 20 25 20 D-xylose 45 40 55 25 25 3-bromo-3-deoxy-D-mannose 5 5 10 10 10 N-acetyl-D-glucoseamine N.D. N.D. 10 10 10 3-bromo-3-deoxy-D-glucose N.D. N.D. 5 N.D. N.D.

All reactions were carried out with 10 mM pyruvate and 50 mM sugar at pH 7.5 and 42°C. The relative rates are tabulated as a percentage of the natural substrate N-acetyl-D-mannoseamine for each of the mutants. N.D., not detectable.

Overall, we have demonstrated the effectiveness of directed exhibit a significant increase in the cleavage activity for L-sialic acid evolution in evolving mutants toward accepting their substrate and an improved condensation of pyruvate and N-acetyl-L- analogues (D- and L-KDO). An efficient L-KDO aldolase was mannoamine as well as several other substrates, providing a pre- created by directed evolution from the E. coli sialic acid aldolase. viously unrecognized route of synthesis to uncommon sugars (Fig. Five rounds of error-prone PCR and iterative screening were 4). For example, D- and L-sialic acid containing a free amine can performed with sampling of 103 colonies per round. The specificity now be efficiently synthesized and further modified for biological constant (kcat͞Km) of the unnatural sugar L-KDO has been im- studies. L-KDO and derivatives can now be efficiently synthesized proved to a level that is equivalent to the specificity constant of the and subsequently used in the mirror-image display selection to wild-type sialic acid aldolase for its natural substrate, D-sialic acid. identify D-peptides and, subsequently, L-peptides for application as The final evolved enzyme (N5B2) exhibits a Ͼ1,000-fold improved antibacterial agents. The mechanistic understanding of enantiose- ratio of the specificity constant [kcat͞Km (L-KDO)]͞[kcat͞Km (D- lectivity could be furthered by evolving the enzyme toward a sialic acid)] and a 30-fold improvement of substrate specificity for complete inversion of enantioselectivity. Development of a more L-KDO over the wild-type aldolase. After the initial two rounds of sensitive method of detection and a higher throughput in vivo analog-guided screening with KDO, improvement of its natural selection system (29) may expedite the progress of directed evolu- enantiomeric counterpart (L-sialic acid) was facilitated with two tion in fine-tuning substrate specificity of aldolases. more rounds of iterative screening with the target substrate, L-sialic acid. The analogue-guided (KDO) strategy overcame an inherent We thank Dr. Ian Wilson and Dr. Andreas Heine for assistance with x-ray crystallography and Dr. Jinq-Chyi Lee, Douglass Wu, and Sheng- difficulty in directed evolution of sialic acid aldolase with an initial Kai Wang for assistance with substrate analogue synthesis and helpful low turnover rate of the natural enantiomeric substrate, L-sialic discussions on molecular modeling. This work was supported by the acid, a rare sugar that is difficult to synthesize in sufficient quantity National Institutes of Health and the Skaggs Institute of Chemical for directed evolution. Two evolved aldolases, N4E6 and N5B2, Biology.

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