1Astbury Centre for Structural Biology, 2School of Biochemistry and Microbiology and 4School of Chemistry, University of Leeds, Leeds LS2 9JT, UK 3Present address: School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA
5 To whom correspondence should be addressed. E-mail: a.berry{at}leeds.ac.uk
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Abstract |
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Keywords: N-acetylneuraminic acid lyase/sialic acid mimetics/structure-guided saturation mutagenesis/synthesis
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Introduction |
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Materials and methods |
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The restriction enzymes EcoRI and HindIII, T4 DNA ligase, Pfu DNA polymerase, WizardTM Miniprep DNA purification kits and DNA markers were obtained from Promega (Southampton, UK), lactate dehydrogenase was from Boehringer Mannheim (Mannheim, Germany), nicotinamide adenine dinucleotide (reduced form) [NADH] lithium salt, N-acetylneuraminic acid (Type IV) and pyruvate were from Sigma Chemicals (Poole, UK), QIAquick and QIAspin DNA extraction kit from QIAgen (Crawley, UK) and Chelating Sepharose Fast Flow from Pharmacia (Milton Keynes, UK). Dialysis tubing was purchased from Medicell International (London, UK). All other chemicals were of analytical grade.
Bacterial strains
E.coli EP-MaxTM 10B Competent cells [(mrr-hsdRMS-mrcBC)
80d lacZ
M15
lacX74 deoR recA1 araD139
(ara, leu)7697 galU galK rpsL nupG
] were supplied by Bio-Rad (UK). The expression vector pKK223-3 was from Pharmacia.
Plasmid construction
The E.coli nanA gene was amplified by polymerase chain reaction (PCR) from genomic DNA and cloned into the expression vector pKK223-3 (Pharmacia) to yield the vector pKnanA. The gene was reamplified from pKnanA using primers NANA-HIS-FOR (5'-TCA GAG GAA TTC ATG GAA CAC CAT CAC CAT CAC CAT GCA ACG AAT TTA CGT-3') and pKK-REV (5'-GGC TGA AAA TCT TCT TCT-3') to add an N-terminal hexahistidine tag to the enzyme to ease purification. The PCR product from the second amplification was subcloned into pKK223-3 using EcoRI and HindIII restriction sites. The ligation mixture was transformed into E.coli EP-MaxTM 10B by electroporation. Plasmid was purified from a single colony and the DNA sequence of the gene was verified. This plasmid, designated pKnanA-His6, was used as the template for mutagenesis.
Saturation mutagenesis at positions Asp191, Glu192 and Ser208
Saturation mutagenesis at positions Asp191, Glu192 and Ser208 was carried out using the Megaprimer PCR method (Reikofski and Tao, 1992). The oligonucleotides used were D191X, 5'-TAT AAC GGT TAC NNK GAA ATC TTC GCC-3'; E192X, 5'-AAC GGT TAC GAA NNK ATC TTC GCC TCT-3'; and S208X, 5'-GGT GGT ATC GGC NNK ACC TAC AAC ATC-3'. In separate reactions, megaprimers were generated for each saturation library using the D191X, E192X and S208X oligos along with pKK-REV and using pKnanA-His6 as template. The PCR reaction was carried out in a 50 µl reaction mixture consisting of 100 pmol each primer, 0.5 µg of plasmid, 0.2 mM each dNTP and 2.5 units of Pfu DNA polymerase in the reaction buffer provided by the supplier. Amplification involved an initial denaturation step at 95°C for 5 min followed by cycling at 95°C for 1 min, 55°C for 1 min and 72°C for 2 min for 30 cycles, then a final extension for 2.5 min at 72°C. The megaprimers were purified from an agarose gel using the QIAspin kit and used as the forward primers in conjunction with pKK-FOR (5'-GGA TAA CAA TTT CAC ACA GG-3') in the next round of PCR, again with pKnanA-His6 as template. Full-length PCR products were purified from agarose gels and subcloned into pKK223-3 as described for the wild-type nanA gene above.
Library screening
Individual colonies were used to inoculate wells of a 96 deep-well plate containing 1.0 ml of 2TY medium with 50 µg/ml ampicillin and 0.1 mM IPTG. The plates were sealed and shaken at 250 r.p.m. at 37°C for 18 h. Following cell growth, 50 µl of each culture were transferred to a 96-well plate containing 50 µl glycerol in each well and stored at 20°C. The remainder was centrifuged at 2000 g and 4°C for 20 min and the supernatants were removed. Each cell pellet was resuspended in 0.5 ml of 50 mM TrisHCl (pH 7.5) containing 1 mg/ml lysozyme. The plates were frozen and thawed at room temperature. Cell debris was collected by centrifugation at 2000 g and 4°C for 20 min and 50 µl of each cleared supernatant were transferred to a new 96-well plate for assay.
The cleavage of screening substrates was monitored by coupling the production of pyruvate to the oxidation of NADH using lactate dehydrogenase. A portion of the cleared supernatants was diluted 4-fold and 50 µl were transferred to a fresh 96-well microtitre plate. To each sample were added 250 µl of 50 mM TrisHCl (pH 7.5) containing 0.33 mM NADH, 0.1 U of lactate dehydrogenase (LDH) and 0.53.0 mM substrate using a multi-channel pipette. The microplate was then placed in a FLUOstar Galaxy 96-well microplate spectrophotometer (BMG Labtechnologies, Aylesbury, UK) and shaken for 10 s (1 mm orbit). The absorbance of each well (after 0.1 s positioning delay) at 340 nm and 30°C was monitored every 60 s for 10 min and the rate of change of absorbance per minute was calculated using the FLUOstar Galaxy software. Selected clones were streaked on to 2TYagarampicillin plates from the frozen glycerol stock plate and grown overnight. Cultures grown from single colonies were used for plasmid purification, DNA sequencing and protein expression.
Protein expression and purification
Glycerol stocks of E.coli EP-Max harbouring plasmids encoding mutant nanA genes were used to inoculate 3 ml of 2TY medium supplemented with ampicillin and grown overnight at 37°C with shaking. These cultures were then used to inoculate 5 l of 2TY medium supplemented with 50 µg/ml ampicillin and 0.1 mM IPTG and grown overnight at 37°C with shaking at 200 r.p.m. Cells were harvested in a Heraeus Contifuge 17RS continual action centrifuge at 19 000 g and then resuspended in 50 ml of 20 mM potassium phosphate buffer (pH 7.5) containing 300 mM NaCl and 10 mM imidazole. The cells were lysed using a French Press at 1500 psi and 4°C. Cell debris was removed by centrifugation at 14 500 g for 1 h at 4°C. Protein was purified using Chelating Sepharose Fast Flow gel charged with Ni2+ using the centrifugation method according to the manufacturer's instructions. Protein was further purified using size-exclusion chromatography. Enzyme was dialysed against 50 mM TrisHCl (pH 7.5) and applied to a Superdex 200 column previously equilibrated with the same buffer. Peak fractions were pooled, concentrated and stored at 4°C.
Enzyme assay
Sialic acid and analogue cleavage was determined by a standard coupled assay with LDH and NADH. The assay was performed in a 1 ml cuvette at 25°C containing 50 mM TrisHCl (pH 7.5), 0.2 mM NADH, 0.5 U of LDH and a suitable aliquot of NAL (1100 µg). The reaction was initiated by the addition of various concentrations of substrate. The decrease in absorbance at 340 nm was recorded as the measure of enzyme activity on a Uvikon 930 spectrophotometer. One unit of lyase activity is defined as the amount of enzyme which catalyses the oxidation of 1 µmol of NADH per minute in this system, using the molar extinction coefficient of NADH as 6220 M1 cm1. Kinetic parameters were estimated by non-linear regression analysis (Cleland, 1979).
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Results |
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Our initial aim was to improve the specificity of NAL towards the production of the dipropylamides 5 (Figure 1), where the 6-glycerol moiety of sialic acid is replaced by a hydrophobic dipropylamide group. These two reactions therefore require different binding pockets in the active site of NAL. Specifically, there is a requirement to provide a favourable environment for the hydrophobic propyl groups whilst simultaneously providing specificity for the new amide group. Random mutagenesis across the entire nanA gene would provide one potential route to the identification of improved variants. However, this approach would require screening at least a few thousand colonies and would require large amounts of the target substrate. This fact, coupled with the availability of several crystal structures of NAL in complex with sialic acid analogues (Barbosa et al., 2000), prompted us to begin with a structure-guided approach. Structure-guided mutagenesis is a useful method for altering the substrate specificity of an enzyme when details of substrate/inhibitoractive site interactions are known (e.g. Wang et al., 2001
; Hill et al., 2003
). The crystal structure of the H.influenzae NAL in complex with three substrate analogues (Barbosa et al., 2000
) allowed the identification of residues thought to be responsible for binding the 6-glycerol moiety of sialic acid (Figure 2A and B). The O8 and O9 hydroxyls of the 4-oxosialic acid inhibitor, and by implication the O8 and O9 hydroxyls of sialic acid, form a bidentate interaction with the carboxylate oxygens of Glu191 (H.influenzae numbering). In addition, O8 also forms a hydrogen bond with the NH of Asp190 and the hydroxyl group of Ser207 forms a hydrogen bond with O7. These three residues (Asp190, Glu191 and Ser207) are strictly conserved in the known NAL sequences from other organisms (Figure 2C).
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Library construction and screening
The equivalent residues in the E.coli NAL enzyme, Asp191, Glu192 and Ser208 (E.coli numbering used from here on), were selected for saturation mutagenesis by MegaPrimer PCR (see Materials and methods). Three mutant libraries, denoted D191X, E192X and S208X, were constructed and protein libraries prepared by lysis of resuspended cell pellets harvested from 1 ml cultures of individually picked colonies from each saturation mutagenesis library. Analysis of randomly selected clones from each library by SDSPAGE showed that 100% of the chosen colonies were expressing protein of similar size to wild-type NAL and that expression levels were not significantly altered (data not shown).
Our aim is to produce an enzyme capable of the synthesis of sialic acid mimetics such as 5. However it is easier to assay the enzyme reactions in the reverse, cleavage (retro-aldol) direction. In this way, successful substrates will yield pyruvate as product, which can in turn be assayed by its reduction with NADH using lactate dehydrogenase. Variants with increased activity in this assay should also show increased activity in the desired synthetic aldol condensation reaction owing to the principle of microscopic reversibility. Similarly, the activity of each variant towards the wild-type substrate, sialic acid, was also measured and the specificity profile of each library is shown in Figure 3. Data in Figure 3 are uncorrected for the level of expression of each variant, although most variants were found to express at approximately the same level as judged by SDSPAGE analysis.
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Activity and substrate specificity of wild-type and mutant NAL proteins
The wild-type N-terminally His6-tagged NAL, and also the chosen variants, were purified by metal chelation affinity chromatography and gel filtration to yield proteins at >95% homogeneity. The steady-state kinetic parameters for the cleavage of sialic acid 3 and the dipropylamide (5a) were then determined (Table I). The dipropylamide is a poor substrate for the wild-type enzyme, being accepted with a specificity constant (kcat/Km) 9-fold lower than that for sialic acid. This ratio of activity agrees well with the ratio determined from assay of the crude cell lysates. The poor specificity constant for the dipropylamide results from both a 3.5-fold lower kcat and a 2.4-fold higher Km for 5a in comparison with sialic acid.
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Clones from substitution at Glu192 produced the greatest improvements in activity with the dipropylamide 5a in the original screening procedure and these improvements were underscored by the steady-state kinetic parameters with pure enzyme (Table I).
All Glu192 variants examined displayed very different substrate specificity to the wild-type NAL. The variant that showed the largest improvement in kcat/Km for the dipropylamide was E192N. This substitution increased kcat for the dipropylamide 1.8-fold and decreased Km 27-fold, resulting in an 50-fold increase in catalytic efficiency with the dipropylamide (as judged by the kcat/Km ratio) compared with wild-type NAL. Concomitantly, the E192N mutant is less efficient with sialic acid as substrate, with a 1.6-fold lower kcat and 8.6-fold higher Km for sialic acid. Hence E192N is 13.4-fold less efficient with sialic acid as substrate than wild-type NAL. As a consequence of these changes in catalytic turnover and Michaelis constant, the E192N variant displays a completely inverted specificity (640-fold) with respect to sialic acid and the dipropylamide, the variant E192N accepting the dipropylamide
6-fold better than wild-type enzyme accepts the natural substrate, sialic acid.
Substitutions of valine and serine at position 192 resulted in similar, but slightly lower, increases in specificity towards the dipropylamide (Table I). Catalytic efficiency towards sialic acid is reduced with both E192V and E192S variants, mainly owing to increases in the Km values for this substrate. In addition to the higher Km value with sialic acid, variant E192V had a 2.2-fold lower kcat, whereas the kcat for the E192S variant was unchanged. With the dipropylamide as substrate, both the E192V and E192S variants showed >30-fold increases in catalytic efficiency; the result of small increases in the kcat of the reaction together with significant decreases in the value of Km for the dipropylamide.
Substitutions of Asp191
Many of the clones from the D191X library showed very low activity with sialic acid and only small improvements in activity with the dipropylamide and only one variant, D191P, was characterized further. Kinetic parameters for the cleavage of sialic acid by D191P could not be accurately determined owing to the very low activity of this mutant. This variant was, however, slightly more efficient with the dipropylamide (1.5-fold) as substrate compared with the wild-type NAL. This is a result of a marginally lower Km (1.9-fold) for the new substrate whereas the kcat for the reaction is not significantly altered. Asp191 is located in the loop between strand g and helix G. The substitution of a proline in this loop might be expected to increase the rigidity of the polypeptide backbone and lead to reductions in kcat for both substrates, as is observed.
Three double mutants (D191E/E192Q, D191E/E192T and D191E/E192M) identified by DNA sequencing of active clones from a screen of a library designed to probe the effect of random changes at position 192 while position 191 was substituted with a Glu were also isolated during the course of this work, but none showed any significant improvements of substrate specificity (Table S1, available as supplementary data at PEDS Online).
Substitutions of Ser208
In general, mutations at Ser208 caused decreases in activity with sialic acid as substrate with only small increases in activity for the dipropylamide. The variant with the highest activity towards the dipropylamide was found to contain a mutation of Ser208 to Gly. Steady-state kinetics of the purified S208G variant showed the same trends in specificity as had been found in the initial screen, but highlighted an inconsistency in the exact magnitude of the specificity change which might be due to the crude nature of the cell extracts used in the initial screen. The purified S208G variant showed a lower Km for the dipropylamide (4-fold lower than the wild-type enzyme), coupled with a
3-fold lower kcat for the reaction. At the same time, the S208G mutant was less efficient with the natural substrate with Km (sialic acid) increased by 2.4-fold compared with the wild-type whereas kcat was not significantly altered. Thus, S208G is only slightly improved with the dipropylamide in terms of catalytic efficiency (kcat/Km) and this mutant remains more efficient with the natural substrate, sialic acid.
Substrate specificity of E192N
The dipropylamide 5a was chosen as the initial screening substrate for these studies because of our hope that engineered active sites which could bind and utilize 5a might also successfully use a range of smaller tertiary amides as substrates. In order to investigate the specificity of the wild-type and E192N mutant NAL towards other 6-carboxamides with various hydrophobic substituents, a number of analogues of 5 were therefore tested as substrates in the coupled enzyme assay and the kcat/Km values determined by steady-state kinetics. A total of seven tertiary amides (including 5) were synthesized as 4:1 mixtures of the C4-equatorial:C4-axial configurations (Woodhall et al., 2005a
) and examined to probe the enzyme specificities. Figure 4 clearly shows that the substrate specificity of the E192N mutant is generally expanded towards tertiary amides in comparison with the wild-type NAL. The E192N mutant displays higher catalytic efficiencies than wild-type NAL with all the carboxamides tested. In fact, E192N is more efficient with five of these substrates than the wild-type enzyme is towards sialic acid. Catalytic efficiency increases with increasing hydrophobicity for the range of substrates tested. For example, the E192N mutant uses the piperidine derivative [(CH2)5] about eight times better than the less hydrophobic pyrrolidine derivative [(CH2)2O(CH2)2]. Similarly, E192N uses the diethylcarboxamide three times less efficiently than the dibutyl derivative. Interestingly, the E192N enzyme is more effective with the dibutyl analogue than with the dipropyl screening substrate 5a, indicating that the size of the engineered active site is not a prime determinant of specificity and suggesting that E192N may be able to tolerate other larger substituents.
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Discussion |
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Glu192 has a major effect on altering the substrate specificity of the E.coli NAL towards the dipropylamide (Table I). This agrees well with its postulated role as a specificity-determining residue within the NAL sub-family, where a glutamate is conserved at that position in all NALs (Figure 2C) but is varied in every other family member except HBHPA. Furthermore, it has been shown that the E188Q mutant of the C.perfringens NAL enhances the cleavage of an alternative substrate, 5,9-di-O-acetyl neuraminic acid, 1.3-fold relative to wild-type NAL (Kruger et al., 2001). Interestingly, it appears that specificity towards the dipropylamide can be increased by substitution of Glu192 with residues which are both polar (Asn and Ser) and non-polar (Val) (Table I). Although the aim of this study was not to delineate the effect of every substitution at position 192, it seems that charge plays an important role in determining specificity at position 192.
Ser208 in NAL is not conserved in any of the sub-family members, but is replaced by an alanine (Ala198) in KDGA. The crystal structure of KDGA (Theodossis et al., 2004), bound with either of the C4-epimeric substrates, 2-keto-3-deoxygluconate (KDG) or 2-keto-3-deoxygalactonate (KDGal), shows that Ala198 interacts with the C5-OH of the product via a water molecule when KDG is bound, but not when the diastereoisomer KDGal is bound. Hence Ala198 may play a role in controlling stereoselectivity in KDGA. By comparison, it may be suggested that Ser208 could play a similar role in NAL and evidence from protein simulations supports this hypothesis (Smith et al., 1999
). However, the results presented here clearly show that alterations of Ser208 do not have a significant effect on the substrate specificity of the enzyme towards the dipropylamides 5.
Overall, our most improved variant, E192N, shows an impressive 50-fold increase in catalytic efficiency with the dipropylamide 5a. Such large increases in efficiency due to single point mutations are rare in protein engineering experiments where whole-gene random mutagenesis is carried out, although some exceptions have been reported. For example, using the E.coli mutator strain XL1 Red, after screening 150 000 colonies, a single amino acid variant of Aspergillus niger amine oxidase was obtained with a kcat 47-fold higher towards the target amine than the wild-type enzyme (Alexeeva et al., 2002). Another example includes the directed evolution of cytochrome P450 BM3 from Bacillus megaterium into a highly efficient catalyst for the conversion of a range of alkanes into alcohols (Glieder et al., 2002
; Peters et al., 2003
). Thus, whereas whole-gene random mutagenesis can yield improvements in activity towards unnatural substrates, saturation mutagenesis at selected residues known to contact the substrate can produce large increases in efficiency while requiring a search of considerably less sequence space. In this way, we have rapidly identified a single mutant of NAL with drastically altered specificity that can now be used as the scaffold for further directed evolution studies. We are currently using a combination of error-prone PCR, saturation mutagenesis and site-directed mutagenesis to fine-tune NAL further towards stereoselective, thermostable biocatalysts for the synthesis of sialic acid mimetics.
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Acknowledgements |
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Received April 11, 2005; accepted April 12, 2005.
Edited by Tony Wilkinson
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