Searching Sequence Space by Definably Random Mutagenesis: Improving the Catalytic Potency of an Enzyme

Proc. Natl. Acad. Sci. USA Vol. 87, pp. 696-700, January 1990 Biochemistry Searching sequence space by definably random mutagenesis: Improving the catalytic potency of an enzyme (evolution/pseudorevertants/spiked oligonucleotides/triose-phosphate isomerase) JEFFREY D. HERMES*, STEPHEN C. BLACKLOW, AND JEREMY R. KNOWLESt Departments of Chemistry and Biochemistry, Harvard University, Cambridge, MA 02138 Contributed by Jeremy R. Knowles, October 20, 1989 ABSTRACT How easy is it to improve the catalytic power mutant triose-phosphate isomerase (D-glyceraldehyde-3- of an enzyme? To address this question, the gene encoding a phosphate ketol-isomerase, EC 5.3.1.1) that has 1/1000th the sluggish mutant triose-phosphate isomerase (D-glyceraldehyde- activity of the wild type. Three questions about the relation- 3-phosphate ketol-isomerase, EC 5.3.1.1) has been subjected to ship between enzyme structure and catalytic power have random mutagenesis over its whole length by using "spiked" been answered. These questions are: (i) can the catalytic oligonucleotide primers. Transformation of an isomerase- activity ofa sluggish enzyme be improved other than by mere minus strain of Escherichia coli was followed by selection of reversion to the wild type (this question is equivalent to those colonies harboring an enzyme ofhigher catalytic potency. asking whether there are other accessible "solutions" to the Six amino acid changes in the Glu-165 -* Asp mutant of triose- catalytic "problem"); (ii) if such improvement were possi- phosphate isomerase improve the specific catalytic activity of ble, would it be rare (that is, are there only a few ways in this enzyme (from 1.3-fold to 19-fold). The suppressor sites are which a protein can accommodate to and compensate for a scattered across the sequence (at positions 10, 96, 97, 167, and particular catalytic lesion); and (iii) how are such second-site 233), but each of them is very close to the active site. These suppressor changes distributed, and can we learn from their experiments show both that there are relatively few single distribution? amino acid changes that increase the catalytic potency of this The recovery of function of a mutant protein by pseudo- enzyme and that all of these improvements derive from alter- reversion is, of course, a well-established phenomenon. The ations that are in, or very close to, the active site. early work of Benzer (11), of Crick et al. (12), and of Yanofsky (13) and later studies [such as those of Sauer and his colleagues on phage A repressor (14)] have shown the There are currently two ways that the manipulation and feasibility of rescuing protein function (15). Yet such inves- mutagenesis of genes that encode interesting proteins can tigations have used methods either where the generation of give new insights into structure-function relationships. In the mutations is not random and the search has been limited by first of these, the presumed importance of a particular amino mutational "hot spots," or where a random search is made acid or of a group of amino acids is probed by changing or only over a limited portion ofthe gene ofinterest. In contrast, deleting it and examining the functional consequences (1-4). we report here the results from a complete and unbiased In its more ambitious forms, this line of attack has led to search across the whole gene that allow us to identify those efforts to use site-specific mutagenesis in a rational way-for alterations that produce catalytic improvement and to rec- example, to produce enzymes of altered specificity (5-7) or ognize those tracts of sequence within which no changes of repressors that recognize a different operator sequence (8, 9). amino acid residues give a more efficient catalyst. These efforts have produced mixed results, however, and it The target enzyme was triose-phosphate isomerase, which is clear that our present understanding of protein structure catalyzes the interconversion of dihydroxyacetone phos- and function does not yet guarantee that rationally designed phate and D-glyceraldehyde 3-phosphate (Fig. 1). The en- changes will yield the predicted outcomes. zyme is an a2-chain dimer of subunit Mr 26,500, and the gene The second and complementary approach does not test our encoding the chicken muscle enzyme has been cloned and current views of the functional consequence of a particular expressed (16) in an Escherichia coli strain (DF502) from structural change but rather aims for a deeper understanding which the endogenous isomerase gene (tpi) has been excised of the problem by identifying all of those structural changes (17). Crystal structures ofboth the chicken (18) and yeast (19) that produce a particular functional effect. Random muta- enzymes alone and in combination with substrate (20) or genesis allows a defined region of mutant sequence "space" inhibitors (21) have been solved to high resolution. Mecha- (10) to be searched by creating a library ofamino acid changes nistic studies (22) have shown that the isomerization pro- in the protein. Selection from this library then identifies ceeds through an enediol(ate) intermediate: Glu-165 acts as mutants that confer the desired phenotype. Since this ap- the base (23, 24), and His-95 (and possibly Lys-13) acts as the proach avoids all preconceived ideas about what is impor- electrophile. The rate constants for every step of the cata- tant, and since (at least in principle) all possible single amino lyzed reaction have been determined both for the wild-type acid changes and some fraction of double and higher order enzyme (25) and for a mutant in which Glu-165 has been changes can be made, the potential exists for the unbiased changed to aspartate (26); for simplicity, this Glu-165 -- Asp discovery of alternative structural responses to a given mutant enzyme is designated "E165D" (single-letter amino functional challenge. In a sense, of course, the random acid code). The wild-type enzyme is an extremely powerful mutagenesis approach mimics the evolutionary refinement of catalyst whose rate of reaction is encounter-controlled (27); biological function at the molecular level. the E165D mutant, in contrast, has a kcatalysis value for In the work reported here, we have identified the single glyceraldehyde phosphate that is smaller by a factor of about amino acid changes that improve the catalytic potency of a 1000 (28). Difference Fourier maps of crystals of these two The publication costs of this article were defrayed in part by page charge *Present address: Merck and Co., Inc., P.O. Box 2000, Rahway, NJ payment. This article must therefore be hereby marked "advertisement" 07065. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 696 Downloaded by guest on September 24, 2021 Biochemistry: Hermes et al. Proc. Natl. Acad. Sci. USA 87 (1990) 697 0 0 H H 0 o OH ©D dihydroxyacetone phosphate D-glyceraldehyde phosphate FIG. 1. The interconversion of dihydroxyacetone phosphate and D-glyceraldehyde phosphate, catalyzed by triose-phosphate isomerase. enzymes show that the only significant change in the struc- (and at least 300,000 transformants from those oligonucleo- ture of the E165D enzyme is a movement of the catalytic tides that encode the known active site functionalities Asp- carboxylate group by about 1 A (E. Lolis and G. Petsko, 165 and His-95). Several sets of transformants were checked personal communication). These findings emphasize the sen- to confirm the efficiency of mutagenesis and the randomness sitive dependence of the reaction rate on the precise posi- of base misincorporation. The transformants from each oh- tioning of catalytic groups. The E165D isomerase mutant, gomer were separately pooled, and the plasmid DNA from fully characterized in both structural and energetic terms, each pool was isolated. Transformation of the isomerase- provides an excellent starting point for catalytic refinement minus E. coli strain (DF502) then allowed the selection of by random mutagenesis and selection. colonies that harbor isomerases having a specific catalytic activity greater than that of the starting E165D enzyme. MATERIALS AND METHODS Selection of Pseudorevertants. Selection for isomerases of Reagents and Strains. Reagents for DNA synthesis were increased specific catalytic activity was performed on M63 obtained from commercial sources for use on a Milligen/ minimal agar plates (33) containing glycerol as the sole carbon Biosearch model 7500 automated DNA synthesizer. Primer- source as described (32). Plasmid DNA from each colony that directed mutagenesis was performed by using the Amersham survived the selection step was then isolated by alkaline lysis oligonucleotide-directed in vitro mutagenesis system. E. coli (34) after growth of an overnight culture, and the isomerase strain DF502 was provided by D. Fraenkel. E. coli strains gene was sequenced across the region of the mutagenic DH5 (29) and TG1 (30), used in the amplification and se- oligonucleotide. Each mutant tpi gene, together with the trc quencing steps, were obtained from commercial sources. promoter used for its expression, was excised on an EcoRI- Plasmid Constructs. pBSRP. A Pvu II fragment containing Pst I fragment, and this fragment was introduced into the the multiple cloning site and part of the lacI and lacZ genes pBSRP vector [a modified version of pBS(+), described (genes encoding respectively the repressor for the f3- above]. Each mutant-bearing plasmid was reintroduced into galactosidase gene and 3-galactosidase) was excised from the isomerase-minus strain DF502, and overnight cultures of pBS(+) (Stratagene Cloning Systems). Oligonucleotide- each mutant were assayed for isomerase activity as described directed mutagenesis (Amersham) was used with a synthetic (32). The isomerases from clones that grew on minimal plates 84-base oligonucleotide to introduce unique EcoRI and Pst I containing glycerol, that gave a higher isomerase activity in sites. crude lysates, and that showed an amino acid change in the pBSTIM.

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