A Combinatorial Approach to Hybrid Enzymes Independent of DNA Homology 1.

Background

Figure 1. DNA Shuffling and Crossover Point (Source: http://academic.pgcc.edu/~kroberts/Lecture/Chapter%207/07-29_Recombination_L.jpg)

DNA shuffling have been used to improve enzyme activity, stability, folding, and to alter substrate specificity. In this technique, parental genes are fragmented and subsequently reassembled by PCR to reconstitute the full-length genes. During this reassembly process, novel combinations of the parental genes arise along with new point mutations. The result of DNA shuffling is a large library of mutant genes from which acquisition of a desired function is selected for using an appropriate selection or screening system. This method require relatively high levels of DNA homology to recombine genes in vitro. However, DNA shuffling cannot exploit alarge portion of the total combinatorial space because crossover points between shuffled genes occur only in regions of relatively high-level DNA homology and at the loci of identity. Crossovers between structurally homologous proteins at sites lacking DNA homology are likely to be productive for protein engineering. Exchange of non-homologous low-energy structures was a more productive strategy than DNA shuffling. However, no combinatorial strategy for creating hybrids between genes that lack DNA homology has been demonstrated. While it is true that DNA shuffling of families of genes with DNA homology can create hybrid enzymes with new properties, such molecular breeding is only feasible for genes with high genetic homology and, for this reason, is unlikely to evolve an entirely novel function. It is important to realize that the primary rationale for success in the shuffling of families of genes is the similarity of the three-dimensional structures of the proteins they encode, not the degree of DNA homology. Indeed, it is an interesting question whether successful directed evolution on homologous families might be equally or better served by the creation of genes with crossovers between family members at regions of little or no genetic homology. Incremental gene truncation libraries can be used to identify loci for the functional bisection of protein and have proposed a number of protein engineering strategies that utuilize incremental truncation. A combinatorial method for biocatalysis engineering called ITCHY (Incremental

Truncation for the Creation of Hybrid Enzymes) creates combinatorial libraries between two genes in a manner that is independent of DNA sequence homology. ITCHY libraries allow the identification of a more diverse set of functional fusions than DNA shuffling. 2. Basic Principle 2.1 Incremental truncation Knowing where to make the fusions is a central problem in the creation of such hybrids. Since current methodologies for genes lacking high homology were limited to `try it and see if it works,' we developed a combinatorial approach to this problem termed incremental truncation. Through incremental truncation we can create fusion libraries of many (or all) different combinations of lengths of two genes. This approach, described herein, is thus a combinatorial solution to the questions `where can enzymes or enzyme fragments be fused to produce active hybrids' as well as `where are the points at which an enzyme can be bisected'. In addition, we outline a method that should circumvent homology limitations to DNA shuffling by allowing shuffling of genes independent of sequence homology. For the average size gene, the separate construction of all possible one-codon truncations would require the assembly of hundreds of plasmids, a labor intensive and time consuming task. Incremental truncation of DNA, on the other hand, allows the construction of a library containing all possible truncations of a gene, gene fragment or DNA library in a single experiment (Figure 2). Incremental truncation is achieved by utilizing the slow, directional, controlled digestion of DNA. During this digestion, small aliquots are frequently removed and the digestion quenched. Thus by taking multiple samples over a given time period we can create a library of all possible single base-pair deletions of a given piece of DNA. We have been using Exonuclease III (Exo III) which exhibits such properties. Exo III has been previously shown to be useful in the creation of large truncations of linear DNA and for techniques in the sequencing of large genes. The digestion rate of Exo III at 37 0C (500 bases/min) is much too fast for purposes of incremental truncation where every one-codon deletion is desired. However, the digestion rate of the exonu- clease can be affected by a variety of methods such as lowering the incubation temperature, altering the digestion buffer composition, inclusion of a nuclease inhibitor or lowering the ratio of enzyme to DNA.

Figure 2. Incremental Truncation (Source: http://www.sciencedirect.com/science/article/pii/S0968089699001431)

Incremental truncation is a method for creating a combinatorial library containing one base pair deletions of a gene or gene fragment of interest. In this protocol, truncations are introduced in opposite directions on fragments from two different genes in two separate reactions. The sets of truncated DNA molecules from each digestion are ligated to each other with DNA ligase. The resulting fusions are cloned as chimeric molecules. The library of cloned fusions is transformed into bacteria and used for further experiments (e.g., phage display, enzymatic activity assay, etc.). 2.2 Hybrid Enzymes Hybrid enzymes are engineered to contain elements of two or more enzymes. A hybrid enzyme is considered to be composed of elements of more than one enzyme. Thus, hybrid enzymes can be generated in a number of ways (Fig. 1): an existing enzyme can be altered by a single point mutation (or series of point mutations) based on structures existing in a second enzyme; similarly, secondary-structural elements or whole domains of enzymes, or monomeric units of multimeric enzymes, can be exchanged; fusions between two enzymes that have separate and distinct activities are also, by this definition, hybrid enzymes.

Figure 3. Generation of hybrid enzymes. (a) Substitution of point mutations, secondary structures or both from enzyme A into a homologous enzyme B. (b) Exchange of functional domains between enzymes C and D or fusion of the intact enzymes. (Source: http://www.jhu.edu/chembe/ostermeier/pdf/04_TrendsBiotech.pdf)

The construction of hybrid enzymes parallels the strategies that nature uses to evolve enzymes. It is generally thought that enzymes have evolved to fit a specific niche in biology through such processes as gene duplication, domain recruitment and fixation of multiple point mutations. Similarly, hybrid-enzyme approaches seek to recruit established functions and properties from existing enzymes and incorporate them into the engineered enzyme. Hybrid enzymes have often been used to determine the differences between related enzymes, identifying those residues or structures that impart a specific property that one enzyme has but another, homologous, enzyme does not. For example, hybrids between two highly homologous proteinases from Lactococcus lactis were used to determine which residues were responsible for their cleavage specificity and rate towards as - and -casein. The hybrids were also used to identify an additional unique domain involved in substrate binding that was absent from related subtilisins. Hybrid enzymes have also been used to investigate the relative merits of structural and sequence alignments between related enzymes.

2.3 Incremental Truncation for Creating Hybrid Enzymes The combination of two incremental truncation libraries called ITCHY creates diversity by fusing two gene fragments. Performing ITCHY on a single gene generates libraries of proteins with internal deletions and duplications whereas performing ITCHY between two different genes generate libraries of fusion proteins in a DNA-homology independent fashion. ITCHY allows the creation f hybrid enzyme libraries between a random length 5fragment of the gene encoding protein A and a random length 3 fragment of the gene encoding protein B. A key step in this process is the digestion of the parent genes with exonuclease III (ExoIII) in the presence of NaCl such that the reaction rate is limited to 10 bases/min. During ExoIII digestion, small aliquots are removed at short intervals and quenched by addition to a low-pH, high salt buffer. As ExoIII digests DNA at a relatively uniform rate, members of the library ostensibly correspond to progressive 1 bp deletions.

Figure 4. Schematic overview of THIO-ITCHY using -phosphothioate nucleotide incorporation by PCR amplification. (a) Linearization of the starting plasmid by restriction digestion at the unique site between the two genes or gene fragments. (b) PCR amplification of the entire linearized vector in the presence of a mixture of dNTPs and S dNTPs as described in Materials and Methods. (c) Incubation of the plasmid with exonuclease III results in hydrolysis of standard dNMPs while the dNMP analogs will block enzymatic degradation. (d) The single-stranded overhangs of the plasmids are removed enzymatically with mung bean nuclease. (e) The blunt-ended constructs are recircularized by intramolecular ligation. (Source: http://nar.oxfordjournals.org/content/29/4/e16/F2.expansion)

3. Case Studies 3.1 Creating interspecies hybrids between E. coli and human genes for GAR tranformylase Gene Gene Function E. coli purN Monofunctional GAR transfomylase of 212 amino acids Human GART segment Trifunctional enzyme glycinamide ribonucleotide synthethase-aminoimidazole ribonucleotide synthetaseglycinamide ribonucleotide transformylase Utilizes cofactor fTHF and is functional as a separate domain

Enzyme Function

Catalyses the transfer of the formyl group from the cofactor N10-formly-tetrahydrofolate (fTHF) to the amino group of GAR to yield formyl-

glycinamide ribonucleotide (fGAR) There is 50% identityat DNA level between the two genes and 41% identity (60% homology) on the amino acid level. Amino acid alignment between purN and the GART segment reveals no gap, although GART lacks nine aminoacids at the C terminus. Structures of the active sites of the two enzymes have been reported to be esentially identical but the structure of GART is not availabel in the Protein Data Bank. 3.2 4. Advantages ITCHY method enabled identification of a more diverse set of active chimeras than DNA shuffling, principally as a result of the relatively nonbiased and non-homology based method that creates the fusions. The active fusions identified by ITCHY demonstrate that crossovers between genes at regions of structural homology, irrespective of DNA sequence homology, are important for creating functional hybrid enzymes. Although the library created by DNA shuffling had a higher frequency of positives, it was not very diverse. Fused genes of ITCHY libraries could have been initially selected for size (e.g. the size of the original genes) resulting in an enrichment for active members of probably 10- to 100fold to give a frequency of 0.1-1.0%. DNA shuffling can create hybrids with multiple crossovers, whereas ITCHY libraries are limited to one crossover point per library member. One can envision an iterative method for ITCHY in order to create library members with multiple crossovers. However, as ITCHY libraries create all possible crossovers between two genes, DNA shuffling of ITCHY libraries should allow one to create a library of genes with multiple crossovers that include crossovers at regions of no homology, thus accessing a more diverse sequence space. In a fashion analogous to DNA family shuffling, which improves directed evolution by accessing a more diverse yet functional sequence space, such a strategy should prove useful for the directed evolution of proteins. In addition, ITCHY libraries should have applications in the creation of novel enzymes by domain and subdomain swapping as well as in the determination of structure/function relationships by characterizing hybrids of interspecies homologs. 5. Improvement An improvement over incremental truncation for the creation of hybrid enzyme (ITCHY) is SCRATCHY (ITCHY combined with DNA shuffling). The approach combines two methods for recombining genes: ITCHY and DNA shuffling. First, ITCHY is used to create a comprehensive set of fusions between fragments of genes in a DNA homology-independent fashion. This artificial family is then subjected to a DNA-shuffling step to augment the number of crossovers. SCRATCHY libraries were created from the glycinamideribonucleotide formyltransferase (GART) genes from E. coli (purN) and human (hGART). References: Nixon, Andrew E. et al. Hybrid Enzymes: Manipulating Enzyme Design. (1998), TIBTECH JUNE 1998 (VOL 16), Elsevier Science Ltd. Nixon, Andrew E. et al. Incremental Truncation as a Strategy in the Engineering of Novel Biocatalysts. (15 October 1998), Bioorganic & Medicinal Chemistry 7 (1999) 21392144. Ostermeier, Marc et al. Combinatorial Protein Engineering by Incremental Truncation. (1999), Proc. Natl. Acad. Sci. USA Vol. 96, pp. 35623567, March 1999 Biochemistry. Dasu, V. Venkata et al. Developments in Directed Evolution for Improving Enzyme Functions. (18 August 2007), Appl Biochem Biotechnol (2007) 143:212223 DOI 10.1007/s12010-0078003-4