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50704/KMO/B583 

MUTANTS OF ENZYMES AND METHODS FOR THEIR USE 

CROSS-REFERENCE TO RELATED APPLICATION(S) 

This application claims the benefit of U.S. Provisional Patent Application 
No. 60/394,886; filed July 10, 2002, the entire disclosure of which is incorporated herein by 
reference. 

FIELD OF THE INVENTION 

This invention relates to novel mutants of leucine dehydrogenase, formate 
dehydrogenase, and galactose oxidase and their applications. 

BACKGROUND 

Unnatural or non-proteinogenic amino acids, which are structural analogs of the 
naturally-occurring amino acids that are the constituents of proteins, have important 
applications as pharmaceutical intermediates. For example, the anti-hypertensives ramipril, 
enalapril, benazapril, and prinivil are all based on L-homophenylalanine; certain second 
generation pril analogs are synthesized from p-substituted-L-homophenylalanine. Various 6- 
lactam antibiotics use substituted D-phenylglycine side chains, and newer generation 
antibiotics are based on aminoadipic acid and other UAAs. The unnatural amino acids L-tert- 
leucine, L-nor-valine, L-nor-leucine, L-2-amino-5-[l,3]dioxolan-2yl-pentanoic acid, and the 
like have been used as a precursor in the synthesis of a number of different developmental 
drugs. 

Unnatural amino acids are used almost exclusively as single stereoisomers. Since 
unnatural amino acids are not natural metabolites, traditional production methods for amino 
acids based on fermentation cannot generally be used since no metabolic pathways exist for 
their synthesis. Given the growing importance of unnatural amino acids as pharmaceutical 
intermediates, various methods have been developed for their enantiomerically pure 
preparation. Commonly employed methods include resolutions by diastereomeric 
crystallization, enzymatic resolution of derivatives, or separation by simulated moving bed 



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EmpreM Mail Na^i^s^^imiZ^^ 



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1 50704/KMO/B583 

(SMB) chirai chromatography. These methods can be used to separate racemic mixtures, but 
the maximum theoretical yield is only 50%. 
5 In the case of non-proteinogenic alkyl straight-chain and branched-chain amino acids 

such as L-nor- valine, L-nor-leucine, L-2-amino-5-[l,3]dioxolan-2yl-pentanoic acid, or L-tert- 
leucine, enzyme-catalyzed reductive amination is an effective method for their synthesis. 
Whereas the naturally-occurring alkyl and branched-chain amino acids can be produced by 
fermentation, taking advantage of the existing metabolic pathways to produce these amino 
acids, stereoselective production of non-proteinogenic analogs and various similar 
compounds is more difficult. The enzyme leucine dehydrogenase has been shown to be 
capable of catalyzing the reductive amination of the corresponding 2-ketoacids of alkyl and 
branched-chain amino acids, and L-tert-leucine has been produced with such an enzyme. 
Improved rates, activity toward a broader range of substrates, and greater enzyme stability 
would offer improved biocatalysts for this type of reaction. It is also an object of this 
invention to describe methods and mutants that can lead to the reductive amination of 2- 
ketoacids to produce D-amino acids such and the D-counterparts of naturally-occurring 
20 amino acids and D-analogs of non-proteinogenic amino acids such as those listed above (D- 
nor- valine, D-nor-leucine, D-2-amino-5-[l,3]dioxolan-2y]-pentanoic acid, or D-tert-leucine). 

Nicotinamide cofactor dependent enzymes are increasingly finding use for the 
synthesis of chirai compounds. Such processes are now in various stages of scale-up and 
2^ commercialization. Aniino acid dehydrogenases are used industrially to synthesize unnatural 
L-amino acids such as L-tert-leucine at the multi-ton scale (Scheme 1). (Kragl et al, 1996) 
Alcohol dehydrogenases have been used to synthesize chirai alcohols, hydroxy esters, 
hydroxy acids, and amino alcohols. An important feature of these reactions is that they are 
chirai syntheses, not resolutions, with yields that can approach 100% of theoretical. The 
starting materials for these types of reactions are the achiral ketones or keto-analogs, which 
are often readily available at low cost. 



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OH + NH4^ L-Amino acid dehydrogenase 



Trimethyl pyruvate 



CO2 
Carbon dioxide 




NADH 



NAD+ 




Formate Dehydrogenase 



L-ferf-Leucine 



JL 



Formate 



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Scheme 1. Synthesis of L-tert-leucine using an amino acid dehydrogenase with formate 
dehydrogenase used for NADH recycHng. 

Because of the relatively high cost of nicotinamide cofactors (in comparison to the 
other starting materials), it is not economically feasible to use the cofactor in stoichiometric 
quantities. Instead, the cofactor must be regenerated in situ using a suitable recycling system. 
The recycling method for the commercial production of L-tert-leucine is based on the use of 
NAD-dependent formate dehydrogenase (FDH) for the regeneration of NADH from NAD+. 
This is an ideal cofactor recycling system because formate is an inexpensive, water-soluble 
reductant, the reaction catalyzed by formate dehydrogenase (formate to CO2) is essentially 
irreversible, and the only byproduct, carbon dioxide, causes no waste disposal or purification 
problems. Furthermore, formate dehydrogenase is now available commercially in bulk 
quantities, as BioCatalytics, Inc. launched the first recombinant form of the enzyme in 2001. 
The commercial formate dehydrogenase enzyme is, however, specific for NAD+ as its 
substrate; it shows no activity toward NADP+. 

Despite the fact that there is no comparable NADP-utilizing formate dehydrogenase 
available, there nonetheless exist a number of extremely useful NADP-dependent enzymes. 
Of particular interest are the NADP-dependent ketoreductases, which catalyze the 
stereoselective reduction of a broad range of ketones to the corresponding chiral alcohols. In 
general, the NADP-dependent ketoreductases catalyze reactions on more complex ketones 



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(those that are also more useful synthetically) than the corresponding NAD-dependent 
enzymes, and ways to exploit their broad catalytic potential are actively being sought. To 
date, we have used glucose dehydrogenase for NADP+ recycling with some success 
(Scheme 2). However, there are certain disadvantages to this. Glucose must be fed as the 
reaction proceeds, and the byproduct, ultimately gluconic acid (from spontaneous hydrolysis 
of gluconolactone) is produced in equimolar quantities and must be separated from the 
desired product. The pH will also drop during this process due to gluconolactone hydrolysis, 
and therefore pH control is necessary. An enzymatic process for the regeneration of NADP+ 
using formate as depicted in Scheme 3 would thus be strongly preferred. 



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p-Keto acid 
or ester 




OH 

Gluconolactone 



Keto reductase 




NADPH 



NADP+ 




Glucose Dehydrogenase 



Ghiral hydroxy 
acid or ester 




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OH OH 



Gluconic acid 



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Scheme 2. Synthesis of a chiral alcohol with the concomitant oxidation of glucose for 
NADPH regeneration. 



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p-Keto acid 
or ester 



CO2 
Carbon dioxide 



Ketoreductase 




NADPH 



NADP+ 




Formate Dehydrogenase 



OH O 



Chiral hydroxy 
acid or ester 



JL 



Formate 



Scheme 3. Proposed synthesis of a chiral alcohol using the mutant NADP-utilizing formate 
dehydrogenase for cofactor recycle. 



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Directed evolution of enzymes is an extremely powerful method to produce new 
enzymes with specific desired properties. In this technique, the gene encoding the enzyme of 
interest is mutagenized and transformed into a host strain such as E. coli to produce a library 
of mutant enzymes. This library, which may contain 5000-20,000 distinct mutants, is 
screened for an enzyme having the desired property. The mutants that test positive for the 
screen can then be subjected to further rounds of mutagenesis and screening in an iterative 
process to obtain an increasingly superior enzyme. This technique has been successfully 
applied to enhance many properties of enzymes including specific activity, thermostability, 
substrate specificity, and enantioselectivity. 

Similar opportunities exist for the use of inexpensive carbohydrate precursors such as 
galactose. The enzyme galactose oxidase converts galactose to the corresponding aldehdye at 
the C-6 position using molecular oxygen as the only co-reactant. Mutants of galactose 
oxidase that are more active, or that act on other carbohydrate or alcohol starting materials, 
would be highly desirable catalysts. 



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SUMMARY OF THE INVENTION 

The present invention is directed to an amino acid sequence that is a mutant of an 
enzyme selected from the group consisting of leucine dehydrogenase sequences as described 
in SEQ ID 2, formate dehydrogenase sequence as described in SEQ ID 1, galactose oxidase 
sequences as described in SEQ ID 3, and substantial equivalents thereof. When the amino 
acid sequence is a mutant of a leucine dehydrogenase sequence as described in SEQ ID 2 or a 
substantial equivalent thereof, the amino acid sequence contains at least one mutation 
selected from the group consisting of F102S, V33A, S351T, N145S and hke mutations in 
subsantially equivalent sequences. When the amino acid sequence is a mutant of a formate 
dehydrogenase sequence as described in SEQ ID 1 or a substantial equivalent thereof, the 
amino acid sequence contains at least one mutation selected from the group consisting of 
D195S, Y196H, K356T and like mutations in subsantially equivalent sequences. When the 
amino acid sequence is a mutant of a galactose oxidase sequence as described in SEQ ID 3 or 
a substantial equivalent thereof, the amino acid sequence contains at least one mutation 
selected from the group consisting of N25Y, T94A, D216N, R217C, M278T, Y329C, 
Q406R, Q406L, V492A, V494A, N521S, N535D, T549I, S567T, T578S and like mutations 
in subsantially equivalent sequences. 

DETAILED DESCRIPTION 

The present invention is directed toward mutant leucine dehydrogenase enzymes, 
mutant formate dehydrogenase enzymes, and mutant galactose oxidase enzymes. In one 
embodiment, the invention is directed to an amino acid sequence that is a mutant of a leucine 
dehydrogenase sequence as described in SEQ ID 2, or its substantial equivalent, with the 
amino acid sequence containing at least one mutation selected from the group consisting of 
F102S, V33A, S351T, N145S and like mutations in substantially equivalent sequences, as 
well as to a deoxyribonucleic acid molecule containing a DNA sequence encoding the 
mutated amino acid sequence. 

In another embodiment, the invention is directed to an amino acid sequence that is a 
mutant of a formate dehydrogenase sequence as described in SEQ ID 1, or its substantial 



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equivalent, the amino acid sequence containing at least one mutation selected from the group 
consisting of D195S, Y196H, K356T and like mutations in substantially equivalent 
5 sequences, as well as to a deoxyribonucleic acid molecule containing a DNA sequence 

encoding the mutated amino acid sequence. 

In another embodiment, the invention is directed to an amino acid sequence that is a 
mutant of a galactose oxidase sequence as described in SEQ ID 3, or its substantial 
jQ equivalent, said amino acid sequence containing at least one mutation selected from the group 
consisting of N25Y, T94A, D216N, R217C, M278T, Y329C, Q406R, Q406L, V492A, 
V494A, N521S, N535D, T549I, S567T, T578S and like mutations in substantially equivalent 
sequences, as well as to a deoxyribonucleic acid molecule containing a DNA sequence 
encoding the amino acid sequence. 

The invention is also directed to a method for the production of an amino acid that 
comprises contacting a ketoacid with an amino acid sequence that is a mutant of the leucine 
dehydrogenase described above in the presence of a reduced nicotinamide cofactor and an 
ammonia source. 

20 The invention is also directed to a method for recycling a nicotinamide cofactor that 

comprises contacting an oxidized nicotinamide cofactor with an amino acid sequence that is a 
mutant of a formate dehydrogenase sequence as described above in the presence of a formate 
source. 

2^ As used herein, the terminology "substantial equivalent" when used to refer to an 

amino acid or nucleic acid sequence encompasses complementary sequences, derivatives, 
analogs, homologs and fragments. 

A nucleic acid molecule that is complementary to a nucleotide sequence shown or 
described is one that is sufficiently complementary to the nucleotide sequence shown such 
that it can hydrogen bond with little or no mismatches to the nucleotide sequences shown, 
thereby forming a stable duplex. As used herein, the term "complementary" refers to Watson- 
Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and 
the term "binding" means the physical or chemical interaction between two polypeptides or 
35 compounds or associated polypeptides or compounds or combinations thereof. Binding 



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includes ionic, non-ionic, Von der Waals, hydrophobic interactions, etc. A physical 
interaction can be either direct or indirect. Indirect interactions may be through or due to the 
5 effects of another polypeptide or compound. Direct binding refers to interactions that do not 

take place through, or due to, the effect of another polypeptide or compound, but instead are 
without other substantial chemical intermediates. 

Moreover, the amino acid or nucleic acid sequence of the invention can comprise only 
jQ a portion of the described amino acid or nucleic acid sequence, e.g., a fragment that can be 
used as a probe or primer or a fragment encoding a biologically active portion. Fragments 
provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 
(contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of 
nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, 
and are at most some portion less than a full length sequence. Fragments may be derived 
from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives 
are nucleic acid sequences or amino acid sequences formed from the native compounds either 
directly or by modification or partial substitution. Analogs are nucleic acid sequences or 
20 amino acid sequences that have a structure similar to, but not identical to, the native 
compound but differs from it in respect to certain components or side chains. Analogs may be 
synthetic or from a different evolutionary origin and may have a similar or opposite 
metabolic activity compared to wild type. 
2^ Derivatives and analogs may be full length or other than full length, if the derivative 

or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or 
analogs of the nucleic acids or amino acid sequences of the invention include, but are not 
limited to, molecules comprising regions that are substantially homologous to the nucleic 
acids or proteins of the invention, in various embodiments, by at least about 45%, 50%, 70%, 
80%, 95%, 98%, or even 99% identity (with a preferred identity of 80-99%) over a nucleic 
acid or amino acid sequence of identical size or when compared to an aligned sequence. 
Alignment can be done manually or using a computer homology program known in the art, or 
whose encoding nucleic acid is capable of hybridizing to the complement of a sequence 
35 encoding the aforementioned proteins under stringent, moderately stringent, or low stringent 



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conditions. See e.g. Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & 
Sons, New York, N.Y., 1993, and below. An exemplary program is the Gap program 
5 (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, 

University Research Park, Madison, Wis.) using the default settings, which uses the 
algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which in 
incorporated herein by reference in its entirety). 

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or 
variations thereof, refer to sequences characterized by a homology at the nucleotide level or 
amino acid level as discussed above. Homologous nucleotide sequences encode those 
sequences coding for isoforms of a polypeptide. Isoforms can be expressed in different 
tissues of the same organism as a result of, for example, alternative splicing of RNA. 
Alternatively, isoforms can be encoded by different genes. In the present invention, 
homologous nucleotide sequences include nucleotide sequences encoding for a polypeptide 
of species other than humans, including, but not limited to, mammals, and thus can include, 
e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide 
20 sequences also include, but are not limited to, naturally occurring allelic variations and 
mutations of the nucleotide sequences set forth herein. Homologous nucleic acid sequences 
include those nucleic acid sequences that encode conservative amino acid substitutions (see 
below) in a polypeptide, as well as a polypeptide having an activity. 
2^ The nucleotide sequence determined from the cloning of one gene allows for the 

generation of probes and primers designed for use in identifying and/or cloning homologues 
in other cell types, e.g., from other organisms, as well as homologs. The probe/primer 
typically comprises a substantially purified oligonucleotide. The oligonucleotide typically 
comprises a region of nucleotide sequence that hybridizes under stringent conditions to at 

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least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand of the 
described nucleotide sequence. 

Probes based on nucleotide sequences can be used to detect transcripts or genomic 
sequences encoding the same or homologous proteins. In various embodiments, the probe 
35 further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a 



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fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part 
of a diagnostic test kit for identifying cells or tissue which misexpress a protein, such as by 
measuring a level of a nucleic acid in a sample of cells. 

The invention further encompasses nucleic acid molecules that differ from the 
described nucleotide sequences due to degeneracy of the genetic code. These nucleic acids 
thus encode the same protein as that encoded by the described nucleotide sequence. 

Accordingly, in another embodiment, an isolated nucleic acid molecule of the 
invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the 
nucleic acid molecule comprising a described nucleotide sequence. In another embodiment, 
the nucleic acid is at least 10, 25, 50, 100, 250 or 500 nucleotides in length. In another 
embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding 
region. As used herein, the term "hybridizes under stringent conditions" is intended to 
describe conditions for hybridization and washing under which nucleotide sequences at least 
60% homologous to each other typically remain hybridized to each other. 

Homologs or other related sequences can be obtained by low, moderate or high 
stringency hybridization with all or a portion of the particular nucleic acid sequence as a 
probe using methods well known in the art for nucleic acid hybridization and cloning. 

As used herein, the phrase "stringent hybridization conditions" refers to conditions 
under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no 
other sequences. Stringent conditions are sequence-dependent and will be different in 
different circumstances. Longer sequences hybridize specifically at higher temperatures than 
shorter sequences. Generally, stringent conditions are selected to be about 5.degree. C. lower 
than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and 
pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid 
concentration) at which 50% of the probes complementary to the target sequence hybridize to 
the target sequence at equilibrium. Since the target sequences are generally present at excess, 
at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be 
those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 
to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 



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30.degree. C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least 
about 60.degree. C. for longer probes, primers and oligonucleotides. Stringent conditions may 
also be achieved with the addition of destabilizing agents, such as formamide. 

Stringent conditions are known to those skilled in the art and can be found in Current 
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the 
conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 
99% homologous to each other typically remain hybridized to each other. A non-limiting 
example of stringent hybridization conditions is hybridization in a high salt buffer comprising 
6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% 
BSA, and 500 mg/ml denatured salmon sperm DNA at 65.degree. C. This hybridization is 
followed by one or more washes in 0.2.times.SSC, 0.01% BSA at SO.degree. C. An isolated 
nucleic acid molecule of the invention that hybridizes under stringent conditions to one of the 
described sequences corresponds to a naturally occurring nucleic acid molecule. As used 
herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule 
having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). 

In another embodiment, a nucleic acid sequence that is hybridizable to the nucleic 
acid molecule comprising a described nucleotide sequence or fragments, analogs or 
derivatives thereof, under conditions of moderate stringency is provided. A non-limiting 
example of moderate stringency hybridization conditions are hybridization in 6.times.SSC, 
S.times.Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 
55.degree. C, followed by one or more washes in l.times.SSC, 0.1% SDS at 37.degree. C. 
Other conditions of moderate stringency that may be used are well known in the art. See, e.g., 
Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY, 
and Kriegler, 1990, Gene Transfer and Expression, a Laboratory Manual, Stockton Press, 
NY. 

In another embodiment, a nucleic acid that is hybridizable to the nucleic acid 
molecule comprising a described nucleotide sequence or fragment, analog or derivative 
thereof, under conditions of low stringency, is provided. A non-hmiting example of low 
stringency hybridization conditions are hybridization in 35% formamide, S.times.SSC, 50 



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mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% FicoU, 0.2% BSA, 100 mg/ml 
denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40.degree. C, followed by 
5 one or more washes in 2.times.SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS 

at 50.degree. C. Other conditions of low stringency that may be used are well known in the 
art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. (eds.), 1993, 
Current Transfer and Expression, a Biology, John Wiley & Sons, NY, and Kriegler, 1990, 
Gene Transfer and Expression, a Laboratory Manual, Stockton Press, NY; Shilo et al., 1981, 
Proc Natl Acad Sci USA 78: 6789-6792. 

In addition to naturally-occurring variants of a given nucleic acid or amino acid 
sequence that may exist, the skilled artisan will further appreciate that changes can be 
introduced into a nucleic acid or directly into a polypeptide sequence without significantly 
altering the functional ability of the protein. In some embodiments, a described nucleotide 
sequence will be altered, thereby leading to changes in the amino acid sequence of the 
encoded protein. For example, nucleotide substitutions that result in amino acid substitutions 
at various "non-essential" amino acid residues can be made in the described sequences. A 
20 "non-essential" amino acid residue is a residue that can be altered from the wild-type 
sequence of without altering the biological activity, whereas an "essential" amino acid residue 
is required for biological activity. For example, amino acid residues that are conserved 
among the proteins of the present invention, are predicted to be less amenable to alteration, 
2^ although some alterations of this type will be possible. 

Another aspect of the invention pertains to nucleic acid molecules encoding proteins 
that contain changes in amino acid residues that are not essential for activity. Such proteins 
differ in amino acid sequence from the described sequences, yet retain biological activity. In 
one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence 
encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% 
homologous to a described amino acid sequence. Preferably, the protein encoded by the 
nucleic acid molecule is at least about 60% homologous to a described sequence, more 
preferably at least about 70%, 80%, 90%, 95%, 98%, and most preferably at least about 99% 
35 homologous to a described sequence. 



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An isolated nucleic acid molecule encoding a protein homologous to a described 
protein can be created by introducing one or more nucleotide substitutions, additions or 
5 deletions into a described nucleotide sequence such that one or more amino acid 

substitutions, additions or deletions are introduced into the encoded protein. Preferably, 
conservative amino acid substitutions are made at one or more predicted non-essential amino 
acid residues. A "conservative amino acid substitution" is one in which the amino acid 
residue is replaced with an amino acid residue having a similar side chain. Families of amino 
acid residues having similar side chains have been defined in the art. These families include 
amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., 
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, 
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, 
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side 
chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, 
phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 
polypeptide is replaced with another amino acid residue from the same side chain family. 
20 Alternatively, in another embodiment, mutations can be introduced randomly along all or part 
of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be 
screened for a desired activity to identify mutants that display that desired activity. 

As used herein, the terminology "like mutations in substantially equivalent sequences" 
2^ refers to mutations in substantially equivalent sequences, as defined above, that are in 
locations different from, but corresponding to, those indicated. For example, deletions or 
insertions can sometimes occur in a nucleic acid or amino acid sequences, creating 
substantially equivalent sequences that are "frame-shifted." These "frame-shifted" sequences 
maintain a similar or homologous sequence of nucleic acids or amino acids except that the 
numerical positions of certain individual nucleic acids or amino acids are shifted to a higher 
number if an insertion of one or more nucleic acids or amino acids has occurred at an earlier 
point in the sequence. Similarly, the numerical positions of certain individual nucleic acids or 
amino acids are shifted to a lower number if a deletion of one or more nucleic acids or amino 
35 acids has occurred at an earlier point in the sequence. 



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As a starting point for the evolving of NADP+ accepting FDH, FDH genes are 
prepared using redesign and synthesis methodology. The gene encoding FDH form Candida 
5 boidini has been redesigned and synthesized to enhance its expression in E. coli. The 

synthesized gene expresses at 20% to 40% of the total protein in the cell, all of which is 
soluble, active enzyme, resulting in formate dehydrogenase. The high level expression of this 
gene in functionally active form enables greater sensitivity in the detection of mutants able to 
accept NADP+ as a substrate. 

Mutagenesis libraries of these genes are prepared using methods developed to create 
mutant genes. Our initial approach focuses on the use of error-prone PGR, such as by error- 
prone PGR protocol described in detail below. This method has been applied to the directed 
evolution of other enzymes, including aminotransferases and alcohol oxidases. We have used 
these methods previously for the generation of mutants of other genes in the successful 
directed evolution of enzyme activities. The method can be fine-tuned as necessary for 
mutagenizing the FDH gene. The mutagenized genes generated as described below are 
transformed into E. coli strain LMG194 or similar for expression and screening. 
20 As a starting point, the template used is the synthetic FDH gene that contains the 

mutation as described by Gul-Karaguler (2001). This gene, designed especially for high-level 
expression in E. coli, is subjected to mutagenesis by error-prone PGR according to a 
modification of the method of May and Arnold [May and Arnold, 2000]. The use of the 
2^ synthetic gene enhances the success of the mutant library by predisposing all derivative genes 
for higher expression in our E. coli host. The error-prone PGR is performed in a 100 piL 
reaction mixture containing 0.25 ng of plasmid DNA as template dissolved in PGR buffer (10 
mM Tris, 1.5 mM MgGb, 50 mM KGl, pH 8.3), and also containing 0.2 mM of each dNTP, 
50 pmol of each primer and 2.5 units of Taq polymerase (Roche Diagnostics, Indianapolis, 
IN USA). The baseline conditions, which can be fine-tuned as necessary, for carrying out the 
PGR are as follows: 2 minutes at 94 ^G; 30 cycles of 30 seconds 94 ""C, 30 seconds 55 °C; 2 
minutes at 72 ""G. The PGR product is double digested with Nco I and Bgl II and subcloned 
into pBAD/HisA vector (Invitrogen, Garlsbad, GA USA) that has been digested with same 
restriction enzymes. The resulting mutant library is transformed into an E. coli host strain 



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LMG194 (Invitrogen, Carlsbad, CA USA) and plated on LB agar supplied with 100 
microgram/mL ampicillin. Individual transformants containing putative mutations are picked 
into 96-well microtiter plates (hereafter referred to as master plates) containing 0.2 mL LB 
Broth with 100 microgram/mL ampicillin, and growth is allowed to take place for 8-16 hours 
at 37 °C with shaking at 200 rpm. Each well in each 96-well master plate is then re- 
inoculated by a replica plating technique into a new second stage 96-well plate pre-loaded 
with the same growth media plus 2 g/L of arabinose, and growth is allowed to continue for 5- 
10 hours at 37 °C with shaking at 200 rpm. The second stage 96-well plates are then 
centrifuged at 4,000 rpm for 10 minutes, and the supernatant is decanted. The cell pellet in 
each well is washed with 200 |iL of water. The washed cell pellet is suspended in 30 (xL of 
B-Per Bacterial Protein Extraction Reagent (Pierce, Rockford, IL USA). Assays are 
conducted using the reduction of NADP+ in the presence formate as an indicator of activity. 
The inventors have found that mutagenesis conditions that produce approximately a 30% kill 
rate (30% of the transformants have inactive enzyme caused by mutations, as assayed against 
the natural substrates) generate 1-3 mutations per gene, and that this rate of mutagenesis is 
useful for creating mutant enzymes with modified activities. 

After the mutants are generated as described above, colonies are picked robotically 
using a colony picker (Autogen, Framingham, MA USA). Up to approximately 2700 
candidate clones can be picked per hour using this colony picker into 96-well (or 384-well) 
microtiter plates. 

Screening is accomplished using a two-stage plating procedure described below for 
96-well plates, but which can be adapted to 384-well plates to increase throughput. Each well 
in each 96-well master mutagenesis plate is re-inoculated by a replica plating technique into a 
new second stage 96-well plate pre-loaded with the same growth media plus 2 g/L of 
arabinose. Growth is continued for 5-10 hours at 37 "^C with shaking at 200 rpm. After 
centrifugation at 4,000 rpm for 10 minutes, the supernatant is decanted, and the cell pellets in 
the second stage 96-well plates are washed with 200 ^iL of water. The washed cell pellets are 
then suspended in 30 jiL of B-Per Bacterial Protein Extraction Reagent (Pierce, Rockford, IL 
USA) for cell lysis. 



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1 



50704/KMO/B583 



After mixing, the suspension of cells in B-Per reagent are allowed to stand for 10 
minutes at room temperature. Then, a solution having the following composition is added to 



1.5 |xL of a 4 mg/mL solution of bromothymol blue indicator 

Wells in which the color changes from blue initially to yellow contain enzymes that are able 
to oxidize formate with NADP+ as a cof actor. These wells are correlated to the original wells 
in the master plates to obtain the original clones of FDH. The sensitivity of the method 
permits the detection of new mutant enzymes having as little as 0.001 micromole per minute 
per milligram of protein, or about 1/1000^*^ the activity of the enzyme on NAD+. 

Background can be reduced by pelleting the cell debris formed by the cell lysis 

20 

procedure, further enhancing the sensitivity of the screen. This additional step is preferably 
implemented only if necessary, as it adds an additional centrifugation operation to the overall 
protocol. 

The best mutants from the first round of mutagenesis described above are reconfirmed 
25 by assay and then sequenced. The mutation or mutations responsible for increased activity are 
determined. Combinations of all different mutations that give rise to increased activity for the 
reduction of NADP+ are prepared and tested to look for synergistic effects of multiple 
mutations in the gene. The best mutants from screening and from the preparation of new 
combinations of synergistic mutations are subjected to further rounds of mutagenesis and 
screening as described above. The further rounds of mutagenesis and screening are carried 
out iteratively to evolve increasingly superior NADP-utilizing FDH enzymes. In general, the 
best 3-5 mutants from each round are carried forward into the subsequent round of 
mutagenesis and screening. 



5 



each well in the plate using a multi-channel pipetting device: 



10 



7.5 |LiL of a pH 8.0 solution containing 8 mg/mL of NADP+ 

7.5 [iL of a pH 8.0 solution containing 0.25 M ammonium formate 

155 ixL of 1 mM potassium phosphate buffer, pH 8.0 



35 



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1 



50704/KMO/B583 



The mutants showing the highest activity from the first and subsequent rounds of 
mutagenesis and screening are reconfirmed by growing cells containing the gene in multiple 

5 1 liter shake flasks. After growth, the cells are harvested, lysed, and the enzyme is purified 

via chromatographic (DE or CM cellulose, or other media) or precipitation (heat treatment or 
ammonium sulfate) methods. SDS-PAGE gels of the crude and purified mutant(s) are taken. 
Kinetic parameters, Vmex, Km (for both formate and NADP+), Kp for NADPH, and pH 

jQ optimum aer determined. The kinetic parameters are preferably determined in two sets of 
experiments. To determine the kinetic parameters of formate, the mutants aer assayed against 
various concentrations of formate (0-100 mM) at a high NADP+ concentration (1 mM). The 
data is fit to the standard Michaelis-Menten equation using nonlinear regression: 



15 



30 



V S 



The kinetic values for NADP+ aer determined in a similar way. The activity is measured at 
20 various NADP+ concentrations (0-1 mM) at a high formate concentration (50 mM). Since the 
cofactor product, NADPH, is known to inhibit FDH, the Kp can also be determined. For this, 
the formate and NADP+ concentrations aer fixed at 50 and 0.5 mM, and the NADPH 
concentration is varied (0-1 mM). The data is fit, using nonlinear regression, to the Michaels- 
Men ten equation modified for product inhibition: 

" 7 V 



i3 



Stability of the new enzymes is measured by incubating the mutant FDH(s) in buffer 
at various temperatures and periodically assaying the enzyme for activity. Stability 
experiments are carried out for 2 half-lives or 1 month, which ever occurs first. 
22 To demonstrate the applicability of the mutant NADP+ accepting FDH, it is used to 

synthesize, on the gram scale, a |}-hydroxy acid or ester. Initially the synthesis of ethyl 4- 



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50704/KMO/B583 



chloro-3-hydroxy butyrate from ethy] 4-chloro-acetoacetate is examined. This is a key 
intermediate in the synthesis of Lipitor^^, with demand exceeding 100 tons per year. The 
inventors have already estabUshed that KRED 1007, one of the novel ketoreductases cloned 
by BioCatalytics, can catalyze the stereoselective reduction of the ketone to produce the S- 
alcohol, which after displacement of chloride by cyanide, is the correct stereochemistry of the 
key C-5 intermediate for further conversion into Lipitor™. The reaction sequence to be used 
is shown in Scheme 4. The net reaction is 4-chloro-acetoacetic ester + formate optically- 
pure S-4-chloro-3-hydroxybutyrate ethyl ester. 



KRED 1007 



Ethyl 4-chloro acetoacetate 




(S)- Eth y 1 4-ch lo ro- 3- hyd roxy 
butyrate 



NADPH 



NADP+ 




CO2 
Carbon dioxide 



Mutant NADP+-accepting 
Formate Dehydrogenase 



L 



Formate 



Scheme 4. Synthesis of (S)-ethyl 4-chloro-3 -hydroxy butyrate using an NADP+-accepting 
FDH to recycle NADPH, 



The procedure used is similar to the biphasic system described by Shimizu et at 
(1990). The substrate and product degrade in water, and therefore a biphasic system is 
necessary as the substrate and product will partition into the organic phase. To 100 ml of n- 
butyl acetate, 6 ml of the ethyl 4-chloro-acetoacetate is added. The enzymes, the mutant FDH 
and a ketoreductase capable of reducing the 2-keto acid to the S-alcohol (BioCatalytics' 
KRED 1007), are added to the aqueous phase (pH 7) to give a total of about 1000 Units each, 
along with NADPh- and formate at 0.15 and 600 mM each, respectively. The two phases are 



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50704/KMO/B583 



mixed thoroughly and the progress of the reaction is monitored via gas chromatography. 
After 100% conversion is obtained, any product in the aqueous phase is extracted into ethyl 
acetate and combined with the butyl acetate phase. The solvent is removed via rotary 
evaporation. Product yield, purity, enantiomeric excess, and total turnover of cofactor aer 
determined. The parameters given above are the starting point and can be adjusted as 
necessary. 

Examples 

The invention will now be described by the following examples, which are presented 
here for illustrative purposes and are not intended to limit the scope of the invention. 

Materials and Sources: 

DNA taq polymerase and T4 DNA ligase can be purchased from Roche Molecular 
Biochemicals (Branchburg, NJ). Restriction endonucleases can be obtained from New 
England Biolabs. The pETlSb expression vector and R coli BL21(DE3) were provided 
previously by Donald Nierlich (UCLA, CA). The pBAD expression vector and E. coli LMG 
194 can be purchased from Invitrogen Corporation (Carlsbad, CA). The cloning vectors 
pGEM-3Z, pGEM-5Zf(-i-)and the host strain E. coli JM109 can be purchased from Promega 
(Madison, WI). Oligonucleotides used for PCR amplification can be synthesized by IDT Inc. 
(Coralville, lA USA) or the University of Florida Core Laboratory (Gainesville, FL USA). 
QlAquick gel extraction kit and QIAprep spin mini-prep kits can be purchased from 
QIAGEN, Inc. (Valencia, CA). DNA sequencing will be carried out by the UCLA DNA 
Sequencing Center (Los Angeles, CA USA) or the University of Florida DNA Sequencing 
Core Laboratory (Gainesville, FL USA). Purification of enzymes can be accomplished using 
Fast Flow DEAE-Sepharose (Pharmacia), CM-celuUose (Whatman) or similar ionic exchange 
materials. Other key enzymes and reagents can be purchased from well-known vendors such 



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50704/KMO/B583 



as Sigma Chemical Company (St. Louis, MO USA), Aldrich Chemical Company 
(Milwaukee, WI USA), VWR (Pittsburgh, PA USA), and the like. 

General Equipment to be used: 

Two SpectroMAX Plus plate readers (accepts both 96 and 384 well plates): Molecular 
Devices Corporation 

Thermocycler for PCR: Perkin Elmer Model 9600 
Deltacycler 11 System: Ericomp 

Shaker/ incubators: Lab-Line and New Brunswick Scientific 
Gel Electrophoresis Apparatus: Bio-Rad and Pharmacia 
Centrifuges: Eppendorf, Beckman, and Sorvall Model RC-3 
Cell lysis: Branson Sonifier 250 and Avestin homogenizer 
Lyophilizer (Aminco) 
Gas Chromatograph (HP-5890) 

HPLC system with diode array detector: Shimadzu VP series with autosampler 
Robotic colony picker: Autogen 

Example 1: Formate dehydrogenase mutants 

Formate dehydrogenase mutants were prepared based on formate dehydrogenase 
having the following native protein sequence (SEQ ED 1): 

MGKIVLVLYDAGKHAADEEKLYGCTENKLGIANWLKDQGHELITTSDKEGETSELD 

KHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYEsrQTGKKISVLE 

VTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIG 

AGRIGYRVLERLLPFNPKELLYYDYQALPKEAEEKVGARRVENEEELVAQADIVTVN 

APLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDV 

WFPQPAPKDHPWRDMRNKYGAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGK 

FDYRPQDIILLNGEYVTKAYGKHDKK. 

Assays of the mutated FDH's were carried out as described above. The following data 
are specific activities with respect to FDH (corrected for %protein and %purity by PAGE). 



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50704/KMO/B583 



All of these activities are measured under saturating conditions (200 mM Formate, 10 mM 
NAD or NADP, pH 7.5, 100 mM KP04, Room Temperature): 

Enzyme NAD Activity NADP Activity 

(U/mg FDH) (U/mg FDH) 

WTFDH 2.2 0.0013 

FDH 1.3 1.5 0.083 

FDH 2.1 1.3 0.19 

FDH 3.1 1.3 0.36 



The mutations are as follows: 

FDH 1.3 D195S 

FDH 2.1 D195S, Y196H 

FDH 3.1 D195S, Y196H, K356T 



Example 2: Leucine Deydrogenase mutants 

Leucine dehydrogenase mutants were prepared based on leucine dehydrogenase 
having the following native protein sequence (SEQ ID 2): 

MGKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPALGGMRMWTYASEEEAI 

EDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRALGRFIQGLNGRYI 

TAEDVGTTVEDMDIIHEETRYVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKEAFG 

DDSLEGKVVAVQGVGHVAYELCKHLHNEGAKLIVTDINKENADRAVQEFGAEFVH 

PDKIYDVECDIFAPCALGAIINDETIERLKCKVVAGSANNQLKEERHGKMLEEKGIVY 

APDYVINAGGVINVADELLGYNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADR 

MAEERIEMMRKTRSTFLQDQRNLINFNNK. 

Four mutants were created and identified through screening that showed enhanced 
activity toward branched chain amino acids L-leucine, L-isoleucine, L-valine, and L-tert- 
leucine. The four mutations were as follows: F102S, V33A, S351T and N145S. Increases in 
activity were from 1.5 to 4 fold relative to the starting wild-type enzyme. 



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50704/KMO/B583 



Example 3: Additional Leucine Deydrogenase mutants 

Through standard molecular biological techniques, all possible combinations of the 
four mutations identified in Example 2 can be created. These mutants can be screened against 
various substrates to establish their catalytic activity for reductive amination or deamination 
reactions. It is also foreseen that other mutations at these positions can be made and screened, 
and that any of these mutations, or combinations of these mutations, can be used in 
conjunction with various silent mutations in the gene. 

Example 4: Galactose Oxidase mutants 

Galactose oxidase mutants were prepared based on galactose oxidase having the 
following native protein sequence (SEQ ID 3): 

MASAPIGSAISRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTFYGANGDPKPPHTY 

TIDMKTTQNVNGLSMLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADST 

TKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPI 

VPAAAAffiFTSGRVLMWSSYRNDAFGGSPGGITLTSSWDPSTGIVSDRTVTVTKHDM 

FCPGISMDGNGQIVVTGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSDGR 

VFTIGGSWSGGVFEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLF 

GWKKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDA 

VKGKILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPD 

GSTFITGGQRRGIPFEDSTPVFTPEIYVPEQDTFYKQNPNSIVRVYHSISLLLPDGRVFN 

GGGGLCGDCTTNHFDAQIFTPNYLYNSNGNLATRPKITRTSTQSVKVGGRITISTDSSI 

SKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNS 

AGVPS V ASTIR VTQ. 

By mutagenesis and screening against aryl alcohol substrates, the following mutants 
of galactose oxidase were created and identified by sequencing. 



-22- 



1 


50704/KMO/B583 




5 


Ref number 


Mutation location 


98 


M278T, V492A, N535D 




110 


N521S, S567T 




112 


R217C , V494A 


10 


146 


R217C, M278T, V492A, N535D 




158 


R217C, M278T, V492A, V494A, N535D 




163 


R217C, M278T, V492A, N521S, N535D 


15 


164 


R217C, M278T, V492A, N535D, S567T 




165 


Q406L 




166 


M278T, Q406L, V492A, N535D, 


20 


176 


R217C, M278T, V492A, V494A, N521S, N535D 


177 


R217C, M278T, Q406R, V492A, N535D 




178 


R217C, M278T, Q406R, V492A, N535D, T549I 




179 


T94A, R217C, M278T, Q406R, V492A, N535D 


25 


180 


N25Y, R217C, M 278T, V492A, N535D, T578S, 




185 


D216N, M278T, Y329C, Q406L, V492A, N535D 




186 


M278T, Y329C, Q406L, V492, N535D 


30 


187 


R217C, M278T, Q406L, V492A, V494A, N521S, N535D 




202 


R217C, M278T, Q406Y, V492A, V494A, N521S, 
N535D, T578S 


35 


203 


R217C, M278T, V492A, V494A, N521S, S, N535D, 
T578S 



-23- 



50704/KMO/B583 



The mutations listed in the table can all be prepared in various combinations by methods 
known to those skilled in the art, creating still additional unique mutants with enhanced aryl 
alcohol oxidase activity. All such mutants are envisioned herein and specifically claimed. The 
individual mutations which may be combined in all possible combinations are as follows: 
N25Y, T94A, D216N, R217C, M278T, Y329C, Q406R, Q406L, V492A, V494A, N521S, 
N535D, T549I, S567T, T578S. It is also foreseen that other mutations at these positions can 
be made and screened, and that any of these mutations, or combinations of these mutations, 
can be used in conjunction with various silent mutations in the gene. 

The preceding description has been presented with references to presently preferred 
embodiments of the invention. Persons skilled in the art and technology to which this 
invention pertains will appreciate that alterations and changes in the described methods can 
be practiced without meaningfully departing from the principle, spirit and scope of this 
invention. Accordingly, the foregoing description should not be read as pertaining only to the 
precise methods described, but rather should be read as consistent with and as support for the 
following claims, which are to have their fullest and fairest scope. 



-24-