NOVEL MODIFIED NAD-DEPENDENT DEHYDROGENASES AND PROCESS FOR THEIR PRODUCTION

Abstract: Novel NAD-dependent dehydrogenases are provided which catalyse the reaction malate <SIGN> oxaloacetate. The enzymes may be further characterised by a low dependence upon the presence of fractose-1,6-biphosphate as activator. The novel enzymes may be desired from wild type enzymes, for example the lactate dehydrogenase of B. stearothermophilus by site-directed mutagenisis.

Description: NOVEL MODIFIED NAD-DEPENDENT DEHYDROGENASES AND PROCESS FOR THEIR PRODUCTION This • invention relates to novel modified NAD-dependent dehydrogenases. Proteins are extremely effective as catalysts for chemical conversions and have evolved to be highly selective. One possibility now emerging from the ability to chemically synthesise genes is the design and construction of proteins with properties not available in nature. NAD-dependent. dehydrogenases are frequently used in assay systems and in stereospecific syntheses of organic compounds. These enzymes catalyse both the hydrogenation of α-keto acids to α-hydroxy acids and the oxidation (dehydrogenation) of α-hydroxy acids to give α-keto acids. The native NAD-dependent dehydrogenase obtained from Baci llus steaτothermophi lus has activity as a lactate dehydrogenase, but has little activity as a dehydrogenase for other substrates. A feature of this particular enzyme is that it is highly thermophilic, i.e. it has a half-life of five minutes at 90°C. There is a need for modified NAD-dependent dehydrogenases, particularly ones which are thermally stable, and which have enzymic activity on selected substrates and/or do not depend on activators. We have now identified residues contributing to the substrate recognition site of NAD-dependent dehydrogenases and shown that the site is present, at least in part, in a particular amino acid sequence. We have further found that modifications to that sequence can result in the formation of NAD-dependent dehydrogenases having, inter alia, different substrate specificities and other commercially desirable properties. To alter or broaden the specificity of an enzyme it is necessary to have a detailed understanding of the mechanism so that the "central" chemistry can be retained.. In the case of enzymes with small substrates, the catalytic and specificity sites are necessarily close together, making it difficult to alter substrate specificity without affecting catalysis. We have now developed a mutation strategy which has enabled the production of NAD-dependent dehydrogenases which have a particularly desirable substrate specificity. In the field of NAD-dependent dehydrogenases, a conventional numbering system has evolved, which was originally based on an X-ray sequence of dog-fish muscle lactate dehydrogenase. This numbering system numbers amino acids in ascending order in a direction extending from, the N-terminus. A discussion of this numbering system is to be found in a paper by Eventoff, W. et al, (1977) Proc. Natl. Acad. Sci. (USA), Vol. 7^, PP. 2677-2681. In this conventional numbering system, amino acid residues in a given polypeptide are not necessarily numbered consecutively from the N-terminus starting with one. Nonetheless there are so-called "essential" and "conserved" amino acids which are always given the same numbers irrespective of the locations at which they appear- (counted from the end terminus). Examples are the two highly conserved glycine residues at positions 30 and 33. the tyrosine at position 85, the arginine at position 109, the serine at position I63 and the aspartic acid at position 168. In a given polypeptide which is a NAD-dependent dehydrogenase (but which is not necessarily a naturally evolved enzyme) there are regions of sequence which are homologous with the amino acid sequence of the conventional numbering system and which enable an alignment of amino acids to be achieved. Such an alignment of amino acid sequences is shewn in the following Table for three NAD-dependent lactate dehydrogenases: the H j , isoenzyme of pig, the testis isoenzyme of man and the B stearothevmcphi lus enzyme. Using similar alignments of their amino .acid sequences, "essential" and "conserved" amino acids of other NAD dependent α-keto acid dehydrogenases may be identified and the numbering correlated with that of the conventional numbering system. In the Table, the symbols "-" do not signify breaks in the contiguous polypeptide chain; instead they are conventional representations of discontinuities of numbering which allow alignment with sequences of other enzymes to give maximum homology. TABLE 1 1 1 2 2 2 2 3 1 0 5 0 3 4 5 0 PIG H A T L K K L I A P v A Q ς E T T I P N N K I T V V G V G - Q V G M MAN X S T V K E Q L I E K L I E D D E - - S Q C K I T I V G T G - A V G M B STEAR M K N N G G A R V V V I G A G - F V G A 3 4 5 5 5 0 5 0 5 PIG H A C A I S I L G K - - S - L T D E L AL V D VL - - ED K MAN X A C A I S I L LK - - D - L A D E LA L V D VA - - L D K B STEAR S Y V F A L M N Q - - G - I - - - - A D E I V L I D A N - - E S K 6 6 7 7 8 8 9 0 5 0 0 0 PIG H L K G E M M D L Q H G S L F L Q T P K I V A N K D Y - S V T A - N MAN X L K G E M M D L Q H G S L F F S T S K V T S G K D Y - S V S A - N B " STEAR A I G D A M D F N H G K V F A P K P V D I W H G D Y - D D C R - D 1 1 1 1 1 9 0 0 1 1 2 5 0 5 0 5 0 PIG H S K I VVVTA G - VRQQ E GES RLNL V QRNVNVFKF I MAN X S RI V I VTAG - ARQQE G ETRLA LVQRNVA IMK I I B STEAR A D LVVIC AG β ANQKP GETR L D LVDKN I AI FRS I 1 1 3 1 1 1 1 2 3 2 2 3 4 4 5 5 0 A B 5 0 5 0 PIG H I P i V Y S P - NC I I I VV S NP VD I L TYVTWKL S G MAN X I P A I VHY S P - D CK I L VV S NP VD I LTY IVWK I S G B STEAR VES VMA S G F - QG L FL VA TNP V D I L TYAT WK F S G 1 1 1 1 1 1 5 6 6 7 7 8 5 0 5 0 - 5 0 PIG H L P K H R V I G S G C N L D S A R F R Y L M A E K L G V H MAN X L P V T R V I G S G C N L D S A R FRY L I G E K L G V H B STEAR - - - - L P H E R V I G S G T I L D T A R F R F L L G E Y F S V A 1 1 1 2 2 1 8 9 9 0 0 9999 0 5 0 5 0 5 A B C D A PIG H S C H w I L G E H G D S S VAVW S G VNVAG V S - L MAN X T C H w I I G E H G D S S VP LW S GVNVAG V A - L B STEAR P QNVHAY I I - GEHGD - TELPVWS QAYI GVMP - I TABLE 1 (contd) 0 1 2 2 3 3 4 B 5 0 5 0 5 0 PIG H Q Q L N P E M G T D N D S E N W K E V H K M V V E S A Y E V I K L MAN X K T L D P K L G T D S D K E H W K N I H K Q V I Q S A Y E I I K L B STEAR R K L V E S K G E E A Q K D - L E R I F V N V R D A A Y Q I I E K 2 2 2 2 2 2 4 5 5 6 6 7 5 0 5 0 5 0 PIG H K - G Y T N - W A I G L S V A D L I E S M L K N - - L S R I H P V MAN X K - G Y T S - W A I G L S V M D L V P - - L K N - - L R R V H P V B STEAR K G A T Y - Y G I A M G L A R V T R A I L H N E N A I L T V 2 2 2 2 2 2 3 7 8 8 9 9 9 0 5 0 5 0 ' 5 9 1 PIG H. S T M V Q G - - - - M Y G I E N E V F L s L P C V L N A R G L T S MAN X S T M V K G - - - - L Y G I K E E L F L s I P C V L G R N G V S D B STEAR S A Y L D G - - - - L Y G E R D - V Y I G V P A V I N R N G I R E 3 3 3 3 3 333 0 1 1 2 2 003 5 0 5 0 • 5 A B 1 PIG H - V I N Q K L K D D E V A Q L K N S A D T L W G I Q K D L K D L MAN X - V V K I D L S E E E - A L L K K S A E T L W N I Q K N L I - F B STEAR - V I E I E L N D D E K N R F H H S A A T L K S V L A R A F T R REFERENCES. MAN X I ϊxpi .es ssion c :1c _n_ Lnc _ of human testis-. « scific lactate dehydrogenase cDNA. Millan.J.L., Driscoll.C.E. and Goldberg E. Unpublished sequence from cDNA (October 1986) B STEAR The DNA sequence of the thermophilic lactate dehydrogenase from Bacillus steaτotheτmophilus . Barstow, D., Clarke, A.R., Wigley, D., Holbrook, J.J. and Atkinson, T. Gene 46 (1986) 47 ~ 55 Within the conventional numbering system, we have identified short sequences which can be correlated with specific structural elements in the folded polypeptide and which may have specific functional properties. For example, they may form the catalytic pathway, the substrate recognition site or the activator site. The substrate recognition site is carried in part by a mobile loop of polypeptide chain, conventionally numbered 98 to 110. This sequence is contiguous but traditionally omits a residue 103- ' The sequence can be identified by the following residues: Gly 106 Glu 107 Ser or Thr 108 Arg 109 Leu 110 Many NAD-dependent dehydrogenases have an activator site which interacts with sugar phosphates and results in the enzyme being dependent for activity at low substrate concentrations on the presence of a sugar phosphate, e.g. fruc ' tose-l t 6-bisphosphate. This dependency results in serious disadvantages when the enzyme is used either in assays or in synthetic organic chemistry. Particularly, if an enzyme having this dependency is used, a sugar phosphate must be included in the reaction medium as an activator and this added'component can complicate subsequent work-up. The activator site (or at least a part thereof) can be identified in a given sequence by examining the sequence for the presence of a sequence of amino acids corresponding substantially to the seven amino acid sequence: Leu.Asp.(Ser or Thr) .Ala.Arg.Phe.Arg which are conventionally numbered 167-173- We have found that the activator site is provided (in part) by this sequence. According to one aspect of the present invention, there is provided a recombinant NAD-dependent dehydrogenase having a basic amino acid at position 102. Examples of basic amino acids include Arg, Lys and His. By "position 102" is meant the position in the conventional numbering system which is six residues to the N- erminal side of the highly conserved arginine at position 109, as in the following representatio :- • • •αα i02- αα 104- αα 105- ctα 106- αα 107' αα 108- Arg - ' ' (n.b. position 103 is conventionally absent). Preferably the recombinant NAD-dependent dehydrogenase has the following amino acids at residues 106 to 110: Gly 106 Glu 107 Ser or Thr 108 Arg 109 Leu 110 It has particularly been found that by modifying a recombinant NAD-dependent dehydrogenase having activity as a lactate dehydrogenase, but having essentially no activity as a malate • dehydrogenase, so as to include at position 102, a basic amino acid, it is possible to produce an enzyme having a higher catalytic activity for malate as substrate than for lactate. Preferably such recombinant NAD-dependent dehydrogenases have an Arg residue at position 102. Preferably the recombinant NAD-dependent dehydrogenases of the invention have the following amino acids: Leu 167 Asp 168 Ser or Thr 169 Ala 170 Arg 171 Phe 172 in their amino acid sequences between residues 167 and 172, and at position 173 have a non-basic amino acid. By "non-basic amino acid" is meant an amino acid other than Arg, Lys or His. Preferably such recombinant NAD-dependent dehydrogenases have a Gin residue at position 173- Recombinant NAD-dependent dehydrogenases may be produced according to the invention which are thermophilic. As such, they are particularly useful as catalytic reagents for industrial purposes as they are stable (thus increasing their useful life) and further they can be used at relatively high reaction temperatures without their decomposition rate becoming excessively high. Consequently they may be used at relatively high temperatures, at which the rate of the required reactions are increased. As used herein, the term "thermophilic" refers to an enzyme which is stable for 20 minutes in lOOmM triethanolamine chloride at pH 6 at a temperature >50 C, - particularly one which is stable for 20 minutes in lOOmM triethanolamine chloride at pH 6 at a temperature >6θ C. The recombinant thermophilic dehydrogenase enzymes of the invention may conveniently possess a substantial amino acid homology with the wild type lactate dehydrogenase of B. stearothermophi lus which is itself thermophilic. According to a further aspect of the invention there are provided recombinant NAD-dependent dehydrogenases consisting essentially of the amino acid sequence of B. stearothermophi lus lactate dehydrogenase and being characterised by having a higher catalytic activity for malate as substrate than for lactate. By "consisting essentially of the amino acid sequence of B. stearothermophi lus lactate dehydrogenase" is meant having major sequence homology at the amino acid level, for example having at least 13% of the amino acids of B. stearothermophi lus lactate dehydrogenase. (See attached Figure, from ref 1). Normally, the sequence homology should be at least 90% and suitably should be at least 98%. Preferably these recombinant NAD-dependent dehydrogenases have a lower dependence for activity at low substrate concentrations on the presence of fructose-1,6-bisphosphate than B. stearothermophi lus lactate dehydrogenase. The invention further provides a method of producing a recombinant NAD-dependent dehydrogenase having a selected substrate specificity, which comprises modifying a cloned DNA sequence coding for an NAD-dependent dehydrogenase so as to form a modified DNA sequence which codes for-a modified NAD-dependent dehydrogenase having a basic amino acid residue at position 102, and expressing the modified sequence in a host. The cloned DNA sequences may be modified by any conventional technique, for example by site-directed mutagenesis. Using the techniques described herein we have constructed a novel malate dehydrogenase from a lactate dehydrogenase framework by changing an amino acid on the mobile coenzyme-loop (residues 98-110) , either alone or in combination with other mutations of the wild-type enzyme sequence. One such change from glutamine to arginine creates a new and optimized binding-site for the oxaloacetate-malate substrate pair and a diminished activity towards the lactate-pyruvate substrate pair. In carrying out the invention, we utilised the DNA which encodes the protein polypeptide chain for lactate dehydrogenases from the thermophilic bacteria, B. stearothermophi lus. The nucleotide sequences of the genes were determined and the DNA, together with regions of sequence both up and downstream of the gene were used to transfect E. co i . The E. colt construction overexpressed lactate dehydrogenase with amino acid sequence, thermal stability and enzyme catalytic properties which were identical to the same enzyme when expressed by the parent thermophilic organism (Refs 1 and 2) . As indicated, the procedure * used to produce our novel constructs was olignucleotide directed mutagenesis. This technique enabled us to produce and isolate in large quantities, enzyme proteins with both single and multiple variations on the original lactate dehydrogenase amino acid sequence. We have also constucted genes coding for hybrid proteins in which a residue, arginine-173 "(which is conserved in all known lactate dehydrogenases) is converted to a non-basic amino acid with the effect of decreasing the sensitivity of the hybrid protein to activation by sugar phosphates, particularly fructose-l,6-bisphosphate. In carrying out the invention, we have isolated the DNA which encodes the protein polypeptide chain for lactate dehydrogenases from thermophilic bacteria, B. stearothermophi lus and B. caldotenax. The nucleotide sequences of the genes were determined and the DNA, together with regions of sequence both up and downstream of the gene were used to transfect E. coli . The E. coli construction over- expressed lactate dehydrogenase with amino acid sequence, thermal stability and enzyme catalytic properties which were ' identical to the same enzyme when expressed by the parent thermophilic organism (refs 1 and 2) . The technique of site-directed mutagenesis, as described in reference 3 was used to specifically alter the amino acid sequence of the original wild-type lactate dehydrogenase enzyme to obtain proteins with new amino acid sequences. As part of this work a mutant enzyme in which an arginine at position 173 o the conventional numbering system was replaced by glutamine was constructed and was shown to have novel and advantageous properties and in particular it was not as dependent upon the presence of the activator fructose-l,6-bisphosphate for high affinity for substrate as the wild-type enzyme. This property enabled the enzyme to be used in enzyme tests and for the conversion of the L-hydroxyacids to the ketoacids without the necessity of adding fructose-1,6- bisphosphate to obtain useful conversion rates and is of commercial advantage. As indicated, the procedure used to produce our novel constructs was olignucleotide directed mutagenesis. This technique enabled us to produce and isolate in large quantities, enzyme proteins with both single and multiple variations on the original lactate dehydrogenase amino acid sequence. Mutagenesis was performed on a 1.3 kb DNA fragment of the B. stearothermophi lus genome containing the let gene. This fragment was subcloned into the EcoRI/PstI sites in the multiple cloning site of M13 mp8 (Barstow, D.A. , Clarke, A.R. , Chia, W.N. , Wigley, D., Sharman, A.F. , Holbrook, J.J. , Atkinson, T. , Minton, N.P. (1986) GENE 46 47-55) • Single-strand phage DNA was prepared from a cell-line transfeeted with this vector and was used as the template for oligonucleotide-directed mutagenesis. 21-base oligonucleotides w ' ere synthesized which were complementary to the template DNA except for the amino acid codons to be mutated. The mutated codon in each case was flanked by 9 identical matches in both the 3' and ' directions. In the Gin 102 -> Arg substitution the GTT codon was replaced by GCC and in the Arg 173 -> Gin substitution the GCG codon was replaced by GTT to give as large a difference as possible in calculated melting temperature over the wild-type sequence. Thnse mismatch oligonucleotides were used as the primers for chain extension with the Klenow fragment of DNA polymerase I. The extended duplex DNA was transfected into a Mut L E. coli strain and plated onto a lawn of TG2. No strand selection procedures were employed. Phage colonies producing mutant DNA were identified by the higher melting temperature of the annealed 5 ** labelled mismatch oligonucleotides. These colonies were plaque-purified and individual plaques were fully sequenced by the dideoxy-nucleotide method to identify both that the desired mutation had been made and that no other had arisen during the manipulations. Double stranded replicative form DNA was prepared from the same culture which yielded the single strand preparation used in sequencing. The EcoRI/PstI fragment was excised from this vector and ligated into the multiple cloning site of the expression vector pKK223-3. The double mutant 102 Gln/173 Arg was constructed by:- (a) digesting the gene coding for the Arg n _ mutant (also referred to as the Q102R mutant) in plasmid pKK223~3 with EcoRI and Sail endonucleases and purifying the 0.4 kbase fragment, (b) digesting the gene coding for the Gin..-,- mutant (also referred to as the R173Q mutant) with Sail and PstI endonucleases and purifying a 0.8 kbase fragment, (c) ligating the 0.4 and 0.8 kbase fragments along with pKK223-3 (pre-digested with EcoRI and PstI) , and (d) transforming E. coli TG2 cells with the recombinant construct. As a result of the above-described transformations we have made a single variation in the sequence of the protein in the active centre in which glutamine at position 102 has been converted to arginine. This enzyme form has a very much enhanced selectivity for the malate/oxaloacetate substrate pair and a very reduced selectivity for the lactate/pyruvate pair. The mutant protein has thus been transformed from one which is a good lactate dehydrogenase to one which is a good malate dehydrogenase and has very poor lactate dehydrogenase activity. The characteristic properties of the newly constructed enzyme are: Pyruvate Oxaloacetate (sec ) (mM) (M sec ) (sec ) (mM) (M sec ) Wild-type 250 0.06 4.2E6 6 1.5 4E3 Construct 1 1.8 550 286 0.06 4.7E6 These properties were measured at 25°C in a support buffer of 0.1M triethanolamine-HCl pH 6 containing 5wM. fructosel,6-bisphosphate. The enzyme was extremely stable and had a half-life at 90°C of 6 mins. The artificial malate dehydrogenase so produced selects against pyruvate (8000:1) to a greater degree than the original lactate dehydrogenase selected against oxaloacetate (1:1000). That is to say, the k /K j . for oxaloacetate is 8,000 times greater than the k 0 ./K J - for pyruvate as a substrate. Thus it is eight times more selected for oxaloacetate than the natural enzyme is for pyruvate. The novel enzyme is twice as active as a natural malate dehydrogenase reported to have been isolated from B. stearot- thermophilus by Murphey et al in 1967 (ref 4). Using the above described transformations, we have further made a single variation in the sequence of the protein in which the . arginine at position 173 is replaced by glutamine. This construct was not as dependent upon the presence of fructose-1,6-bisphospahate for high affinity for substrate as the wild type enzyme. A further construct has been prepared with both mutations, i.e it contains a new malate dehydrogenase active centre and has had the fructose-1,6-bisphosphate site removed. This enzyme is an active malate dehydrogenase and also it is less sensitive to fructose-1,6- bisphosphate. This enzyme has particular advantages for use in enzyme test kits and in enzyme reactors. REFERENCES (1) Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene. Barstow D; Clarke AR; Chia WN; Wigley D; Sharman AF; Atkinson T; Minton NP; Holbrook JJ Gene (1986) 4647-55 (2) Amino acid sequence of the L-lactate dehydrogenase of Bacillus caldotenax deduced from the nucleotide sequence of the cloned gene Barstow D; Murphy JP; Sharman AF; Clarke AR; Holbrook JJ; Atkinson T; Eur. J. Biochem. (1987) 165581-6 (3) Site-directed mutagenesis reveals the role of a mobile arginine residue in lactate dehydrogenase catalysis. Clarke AR; Wigley DB; Chia WN; Barstow D; Atkinson T; Holbrook JJ; Nature (1986) 324 699-702 (4) k 125s for a B. stearothermophilus malate dehydrogenases on a different protein framework isolate in 1967 Murphey WH; Barnaby C; Lin FJ; Kaplan NO; (1967) J. Biol. Chem. 2421548-59)

Claims: CLAIMS 1. A recombinant NAD-dependent dehydrogenase having a basic amino acid at position 102. 2. A recombinant NAD-dependent dehydrogenase according to claim 1 wherein said basic amino acid is selected from Arg, Lys and His. 3. A recombinant NAD-dependent dehydrogenase according to claim 1 or claim 2 having the following amino acids at residues 106 to 110: Gly 106 Glu 107 Ser or Thr 108 Arg 109 Leu 110 4. A recombinant NAD-dependent dehydrogenase according to any preceding claim having a higher catalytic activity for malate as substrate than for lactate. 5. A recombinant NAD-dependent dehydrogenase according to any preceding claim having an Arg residue at position 102. 6. A recombinant NAD-dependent dehydrogenase according to any preceding claim having in its amino acid sequence between residues 167 and 17 , the.following amino acids: Leu 167 Asp 168 Ser or Thr 169 Ala 170 Arg 171 Phe 172 and at position 173 non-basic amino acid. 7. A recombinant NAD-dependent dehydrogenase according to claim 6 having a Gin residue at position 173- 8. A recombinant NAD-dependent dehydrogenase consisting essentially of the amino acid sequence of B. stearothermophilus lactate dehydrogenase and being characterised by having a higher catalytic activity for malate as substrate than for lactate. 9. A recombinant NAD-dependent dehydrogenase according to claim 8 consisting of at least 757» of the amino acids of B. stearothermophilus lactate dehydrogenase. 10. A recombinant NAD-dependent dehydrogenase according to claim 8 or claim 9 having a lower dependence for activity at low substrate concentrations on the presence of fructose-1,6-bisphosphate than B. stearothermophi lus lactate dehydrogenase. 11. A method of producing a recombinant NAD-dependent dehydrogenase having a selected substrate specifity, which comprises modifying a cloned DNA sequence coding for an NAD-dependent dehydrogenase so as to form a modified DNA sequence coding for a modified NAD-dependent dehydrogenase having a basic amino acid residue at position 102, and expressing the modified sequence in a host.

Inventors: ATKINSON ANTHONY, CLARKE ANTHONY R, HOLBROOK J JOHN, ATKINSON, ANTHONY, CLARKE, ANTHONY, R., HOLBROOK, J., JOHN
Applicants: HEALTH LAB SERVICE BOARD, UNIV BRISTOL, PUBLIC HEALTH LABORATORY SERVICE BOARD, THE UNIVERSITY OF BRISTOL
Application year: 1989
Main classification: C12N15
IPC codes: C12N9, C12N15, C12R1