Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution

Stuart J. Allen1 and J.John Holbrook

Molecular Recognition Centre, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk,Bristol BS8 1TD, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacillus stearothermophillus lactate dehydrogenase (bsLDH) is activated in the presence of fructose 1,6 bisphosphate (FBP). The activator is expensive and representative of the sort of co-factor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant (6A) which is almost fully activated in the absence of FBP. Wild-type bsLDH has a KpyrM of 5 mM in the absence of FBP but when activated (+FBP) the KpyrM drops to 0.05 mM. The mutant 6A has a KpyrM of 0.07 mM in the absence of FBP. 6A has three amino acid substitutions—R118C, Q203L and N307S—resulting in a 70-fold activation, none of the mutations are near the active site. The activation of wild type bsLDH is due to an FBP induced tetramerization of dimeric bsLDH bringing about a structural rearrangement of key active site residues. The most likely explanation for the activation of 6A is derived from the position of Q203L, which is at the dimer–dimer interface. The suggestion is that the hydrophilic to hydrophobic change has altered the dimer–tetramer equilibrium position towards that of the tetramer. What is significant is the activation of bsLDH by a subtle long range event produced by the `blind' directed evolution approach.

Keywords: directed evolution/fructose 1,6 bisphosphate/lactate dehydrogenase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The chemoenzymatic synthesis of fine chemicals is a rapidly expanding area. There are twin pressures being exerted on the chemical industry in the form of an ever increasing demand for the `greening' of chemical manufacture as well as a shifted emphasis towards the production of single isomer pharmaceuticals. Biocatalytic routes for the production of high margin enantiopure pharmaceutical and agricultural intermediates look increasingly attractive.

Qualms over the use of enzymes in chemical manufacture have traditionally centred on the instability and complexity of biological catalysts. Thermostable enzymes are robust, have good general stability and display a reasonable degree of tolerance to organic solvents. Reactions can be run at higher temperatures which eases solubility, viscosity and microbial contamination problems. Thermostability also allows simpler upstream processing. The range of catalytic activities of enzymes is impressive but often not optimized to exact industrial requirements. It is important to be able to quickly tailor an enzyme to a needed function. Much work has focused on the hydrolytic enzymes and their applications to chiral resolutions. Dehydrogenases show significant promise for pharmaceutical manufacture as they introduce chirality into a prochiral centre and as such may constitute a key step in any synthesis.

Bacillus stearothermophillus lactate dehydrogenase (bsLDH) is a thermostable L-2-hydroxyacid dehydrogenase that is allosterically activated by the expensive co-factor fructose 1,6 bisphosphate (FBP). The non-activated form has a KM for pyruvate of 5 mM but in the presence of FBP the KM for pyruvate drops to 0.05 mM, a 100-fold improvement (Clarke et al., 1985aGo,bGo). Activation of bsLDH is due to an FBP induced tetramerization of dimeric bsLDH that brings about a structural rearrangement of key active site residues (Clarke et al., 1987Go). It has proved difficult to engineer out FBP activation by rational design methodologies as significant yet subtle and multiple structural rearrangements are required.

Previously the substrate specificity of bsLDH has been very successfully altered by a semi-rational approach (El Hawrani et al., 1996Go) where two positions known to be associated with determining substrate specificity were randomized and the resulting mutants screened. We were interested to see if this system could be pushed further into a random mutate and screen process to solve a problem that for the reasons outlined above knowledge based approaches have proved difficult. We carried out rounds of mutagenesis and screening in the absence of FBP under successively reduced substrate concentrations in an attempt to improve bsLDH substrate specificity in the absence of FBP.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and oligonucleotides

Escherichia coli TG2, supE hsd{Delta}5 thi{Delta}(lac-proAB) {Delta}(srl-recA)306:: Tn10(tetr) F' [traD36 proAB+ lacIq lacZ{Delta}M15] was used throughout. Plasmid pKK223-3 (Pharmacia) was used for all standard cloning procedures and for protein expression. Two oligonucleotides were used for PCR: LDH-NTER, 5'-CGGAATTCATGAAAAACAACGGTGGAGC-3'; LDH-CTER, 5'-AAAACTGCAGTCATCGCGTAAAAGCACG-3'.

Construction and mutagenesis of target pLDH2

The pLDH2 target construct consisted of the ORF of Bacillus stearothermophillus L-lactate dehydrogenase (bsLDH) (Barstow et al., 1987Go), minus flanking sequences, cloned into the expression vector pKK223-3. The ORF of bsLDH was amplified by PCR using the LDH-NTER and LDH-CTER primers. The resulting fragment was EcoRI/PstI-digested and cloned in suitably restricted pKK223-3. Random mutagenesis of pLDH2 was done by the DNA shuffling method (Stemmer, 1994Go). An approximately 1 kb fragment containing the entire bsLDH gene was produced by PCR using the LDH-NTER and LDH-CTER primers. The PCR mix was 10 ng pLDH2 template, 200 µM each dNTP, 1 µM each primer LDH-NTER and LDH-CTER, 5 µl 10x Taq buffer and 2.5 U Taq DNA polymerase (Boehringer Mannheim). The reaction was carried out for 25 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s in a Perkin Elmer thermal cycler. The 1 kb fragment was run on a 1% agarose gel and purified using the Qiaquick gel extraction kit (QIAGEN). The 1–2 µg of target DNA was digested with 0.0002 U DNase I for 25 min at 20°C in a 100 µl reaction containing 50 mM Tris–HCl (pH 7.5), 1 mM MgCl2, 0.1 mg ml–1 BSA. 100–200 bp fragments were gel purified and reassembled by PCR in a mix that contained 200 µM each dNTP, 5 µl 10x Taq buffer and 2.5 U Taq DNA polymerase in 50 µl. The PCR was done for 25 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 30 s. Full-length product was amplified from 2 µl of the reassembled mix with 200 µM each dNTP, 1 µM primers LDH-NTER and LDH-CTER, 5 µl 10x Taq buffer and 2.5 U Taq DNA polymerase, 25 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s. The resulting 1 kb product was EcoRI–PstI digested and cloned back into pKK223-3. The typical ligation reaction consisted of ~40 ng insert, ~50 ng vector, 1x T4 DNA ligase buffer and 0.5 U T4 DNA ligase in a total volume of 20 µl. The resulting pKK-LDH construct was electroporated into E.coli TG2 cells and selected on 2x YT agar containing 100 µg/ml ampicillin.

Screening

The activity screen was essentially as described previously (El Hawrani et al., 1996Go). Colonies were lifted onto nitrocellulose filters and dried briefly on 3MM Whatman paper at room temperature. 3MM Whatman sheets were cut to fit 20x20 cm plastic trays and soaked in buffer I (10 mM Tris–HCl pH 7.4, 1 mM EDTA) containing 4 mg/ml chicken egg white lysozyme (EC 3.2.1.17). The nitrocellulose filters were placed onto the lysozyme-soaked Whatman and incubated at 30°C for 30 min. The filters were then removed to dry Whatman for a few minutes and washed by being placed on Whatman soaked in buffer I for 5 min. The filters were transferred back to dry Whatman for a few minutes and a second lysis step conducted on Whatman soaked in buffer I plus 1% (v/v) Triton X-100 incubated at 37°C for 5 min. The filter was dried and washed in buffer I as before, placed onto fresh Whatman soaked in buffer I and incubated at 60°C for 30 min. The filters were finally transferred to Whatman soaked in assay buffer II (Tris–HCl pH 8.0). The assay mix containing 1 mM NAD+, 1 mg/ml nitroblue tetrazolium, 0.5 mg/ml phenazine methosulphate and 10–40 mM lactate made up in buffer II and sprayed onto the filters. The filters were incubated in the dark for 30–60 min and bsLDH containing colonies with activity under the given conditions showed up as blue halos around the colony.

Protein purification

Mutant and wild-type bsLDH were purified by a two step procedure. Overnight cultures (1 l) were centrifuged at 1900 g, and the pelleted cells resuspended in 50 mM triethanolamine (pH 7.5). The cells were sonicated and the debris removed by centrifugation at 27 000 g. The supernatant was subjected to a heat step of 65°C for 30 min and the denatured material pelleted by centrifugation at 27 000 g. After dialysis in 50 mM triethanolamine, NADH was added to the protein to a final concentration of 10 mM, before loading onto an oxamate-Sepharose column which had been pre-equilibrated with 50 mM triethanolamine (pH 6.0), 0.5 mM NADH. Unbound protein was eluted with 50 mM triethanolamine (pH 6.0), 0.3 M NaCl, and LDH eluted with 50 mM triethanolamine (pH 9.0), 0.3 M NaCl. The bsLDH-containing fractions were pooled and precipitated with 65% saturation ammonium sulfate. The precipitated protein was resuspended in 20 mM triethanolamine (pH 6.0) and dialysed against the same buffer containing 1 mg/ml activated charcoal.

Enzyme kinetics

Steady-state kinetic measurements were made by following the decrease in A340 due to NADH consumption. Data was analysed using non-linear regression analysis programs within GRAFIT (Leatherbarrow, 1989Go). Assays were conducted in 20 mM Bis-Tris (pH 6.0) at 20°C. Enzyme concentrations were typically 5–35 nM.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Screening for improved bsLDH activity in the absence of activator (FBP)

DNA shuffling was used to create a bsLDH library of 3–4 base changes per gene (~0.4% mutation rate). This rate was desired as it should, on average, create a library of single amino acid variants which it was felt would offer sufficient diversity while limiting the accumulation of detrimental mutations. In the screen used here E.coli expressing recombinant random mutants of bsLDH were transferred to nitrocellulose filters, the cells were lysed and the protein made to diffuse away from the cell debris (Figure 1AGo). Activity was detected by spraying the filters with a PMS/NBT containing cocktail (Figure 1BGo) which produces a blue halo around the active colonies. After the first round of mutagenesis 214/311 (69%) colonies showed activity, blue halos, in the screen when assayed with 40 mM lactate in the absence of FBP. From this round 17 colonies were picked at random and analysed by SDS–PAGE, 11/17 (65%) were correctly expressed, folded and heat stable which correlated well to the filter assay results.



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Fig. 1. (A) Mutagenesis and screening methodology used in the production of mutants of Bacillus stearothermophillus lactate dehydrogenase. (B) Schematic of the PMS/NBT assay used to look for activity in dehydrogenase mutants. The methodology and assay is broadly applicable to thermostable dehydrogenases.

 
Round 1 was produced again, as before screening at 40 mM lactate in the absence of FBP, which resulted in 831/990 (84%) positives from which the best 20 as judged by eye were picked. Round 2 was done at 20 mM lactate in the absence of FBP from which the best 20 colonies were picked. The final round was assayed at 10 mM lactate (–FBP) from which six bright colonies were identified. Of these one (6A) showed strong activation in the absence of FBP (Table IGo).


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Table I. Steady-state kinetics constants for wild-type bsLDH and the mutant 6A
 
Kinetics of activated (6A) mutant

The mutant 6A is almost fully activated in the absence of FBP; this represents a 70-fold improvement in pyruvate affinity in the absence of FBP compared with wild-type bsLDH. 6A shows substrate inhibition in the absence of FBP which is normally only seen in the activated (+FBP) form of wild-type bsLDH. Thermostability though has been slightly affected, 6A shows 70% activity retained after 30 min at 65°C compared with 100% in wild-type bsLDH under the same conditions. 6A shows an approximately threefold reduced catalytic rate compared with unactivated wild type but a rate ~70% the catalytic rate of the activated (+FBP) form of wild-type bsLDH.

Sequence analysis

Determination of the DNA sequence of the mutant 6A has shown four base changes resulting in three amino acid substitutions: R118C, Q203L and N307S.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mutant 6A was obtained after three rounds of mutagenesis and selection, of 2000–3000 colonies per round, under increasingly reduced substrate conditions in the absence of the activator fructose 1,6 bisphosphate. The total number of colonies screened is estimated to be <10 000 at a mutation rate of 0.4% (1 base in 250 mutated) per generation. This rate of mutation should give, on average, one amino acid substitution per generation. In the first round, 84% of the colonies were active, correctly folded and heat stable, by the third round only 33% (4/12) of the colonies were active, correctly folded and heat stable. 6A, which was isolated in the third round, has three amino acid substitutions: R118C, Q203L and N307S.

None of the substitutions are near the active site of the enzyme; however, from previous studies on bsLDH (Clarke et al., 1987Go) along with the position of the mutations as assigned by reference to the wild-type bsLDH crystal structure in its teterameric (+FBP) form (1LDN) a probable explanation can be proffered. R118C lies along the centre of helix {alpha}D-{alpha}E pointing into the solvent. N307S is a conservative change which is positioned in a loop region just before helix {alpha}H again pointing into the solvent. Q203L lies at the end of {alpha}H at the dimer–dimer interface. In the bsLDH tetrameric crystal structure Q203L points at an `oily' patch (V208, M209) on a loop of the opposite dimer. If this is the case, then as each mutated residue of the LDH monomer interacts with its opposite loop in the tetramer four hydrophobic interactions will be made. Thus a single amino acid replacement (Q203L) results in four interactions per tetramer which along with the increased energy required to solvate the exposed leucine in the dimeric form is sufficient to significantly shift the dimer– tetramer equilibrium towards that of the tetramer. The assumption is that the multiple contacts at the dimer–dimer interface brings about the necessary structural rearrangements in the active site of the enzyme to produce the tight binding form of bsLDH.

The kinetics bears out the above in that substrate inhibition occurs only in the tight binding tetrameric form (+FBP) of wild-type bsLDH accompanied by a reduction in the catalytic rate. The same kinetic characteristics is seen in the mutant 6A in the absence of FBP. Finally, 6A has maintained its thermostable qualities, it is slightly less stable than wild-type bsLDH, at 65°C for 30 min it has 75% of the residual activity of wild type. However, this may be due to one of the mutations other than Q203L, a likely candidate being R118C, which may now be able to form intermolecular disulphides. We are currently dissecting the roles of the various mutations.

We aimed to test the viability of a `blind' directed evolution approach to a problem that had proved refractory to rational design. We have produced an enzyme that no longer requires the activator FBP. This required significant and subtle structural rearrangements whilst maintaining thermostability which has been found by screening a relatively small number of mutant proteins.


    Notes
 
1 To whom correspondence should be addressed;email: S.J.Allen{at}bristol.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barstow,D.A., Murphy,J.P., Sharman,A.F., Clarke,A.R., Holbrook,J.J. and Atkinson,T. (1987) Eur. J. Biochem., 165, 581–586.[Abstract]

Clarke,A.R., Atkinson,T., Campbell,J.W. and Holbrook,J.J. (1985a) Biochim. Biophys. Acta, 829, 387–396.[ISI]

Clarke,A.R., Waldman,A.D.T., Hart,K.W. and Holbrook,J.J. (1985b) Biochim. Biophys. Acta, 829, 397–407.[ISI][Medline]

Clarke,A.R., Atkinson,T. and Holbrook,J.J. (1987) Trends in Biochem Sciences, 14, 101–105.

El Hawrani,A.S., Sessions,R.B., Moreton,K.M. and Holbrook,J.J. (1996) J. Mol. Biol., 264, 97–110.[ISI][Medline]

Leatherbarrow,R.J. (1989) GRAFIT. Erithacus Software Ltd, Staines, UK.

Stemmer,W.P.C. (1994) Nature, 370, 389–391.[ISI][Medline]

Received August 3, 1999; revised October 18, 1999; accepted October 18, 1999.





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