Hybrid enzymes: manipulating enzyme design

Nixon, Andrew E.; Ostermeier, Marc; Benkovic, Stephen J.

doi:10.1016/s0167-7799(98)01204-9

Cited by 81 publications

(43 citation statements)

References 56 publications

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“…The exchange of functional domains between existing proteins is one way to obtain hybrid enzymes with desired activities and properties (25). To date, the construction of hybrid endonucle- ases using this approach has been reported for several groups of these proteins.…”

Section: Discussionmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.hybrid ͉ AloI ͉ PpiI ͉ TstI R estriction endonucleases (REases) are parts of restrictionmodification (R-M) systems, whose primary biological function is the protection of bacterial cells from incoming foreign DNA molecules (1). There are three main groups of restriction enzymes (types I, II, and III), which differ in enzyme composition, cofactor requirements, and mode of action (2). The beststudied are type II REases, which in general recognize specific DNA targets of 4-8 bp and cleave DNA at or close to these sequences (1, 2). The exquisite accuracy of type II enzymes (3, 4) has made them indispensable tools for DNA manipulations. Although almost 3,700 type II REases with 262 different specificities have been characterized to date (5), there still is a demand for enzymes recognizing new DNA targets.During the past two decades, numerous efforts have been undertaken to engineer type II REases with altered specificities. Both rational protein design and random mutagenesis, followed by various selection procedures, have been tried (6), and several mutant enzymes with some preference for cleavage of altered DNA targets were isolated (7-10). However, projects concerned with orthodox type II REases so far have been largely unsuccessful mainly for two reasons: (i) difficulty of dealing with the observed tight coupling between DNA recognition and cleavage and (ii) absence of an efficient system for selecting enzymes with changed specificities. In this regard, unorthodox type II enzymes, such as the type IIG REase Eco57I (11), have shown more promise. Type IIG enzymes combine the catalytic centers of endonuclease and methyltransferase in one polypeptide chain, and the ability of Eco57I to methylate recognized DNA targets has been applied to isolate mutants having previously undescribed specificity (12).The discovery of AloI-like REases (13-15), classified as type IIB enzymes, opened up new opportunities for the engineering of type II REases with altered specificities. AloI-like REases ar...

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Section: Discussionmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, functional fusion of two enzymes was expected to have several advantages over individual enzymes with respect to reaction kinetics and enzyme production, as well as novel properties and reactivity. As practical cases of the multistep sequential reaction, the performance of the hybrid or fusion enzymes sometimes was better than that achieved by successive action of individual enzymes, expanding the potential use of natural enzymes (6,21,25).Previously, we cloned and expressed two hydantoinase genes from Bacillus stearothermophilus SD1 (17) and Bacillus thermocatenulatus GH2 (15). We also identified some conserved domains possessing essential amino acid residues by comparative analyses of functionally related enzymes (16).…”

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confidence: 99%

“…In the field of molecular biology and biotechnology, enzymes possessing two or more combined activities, along with appropriate stability, have found wide application (25). Although the natural diversity of the enzymes provides some candidates that have evolved to possess bifunctional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes based on evolutionary traits and well-defined structure (1,25).…”

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confidence: 99%

“…Although the natural diversity of the enzymes provides some candidates that have evolved to possess bifunctional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes based on evolutionary traits and well-defined structure (1,25). Artificial fusion enzymes, simply generated by either end-to-end fusion or by tethering of whole genes encoding intact functional proteins with a linker, have been reported to show noticeable performance in a concerted fashion (6,21).…”

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Construction and Evaluation of a Novel Bifunctional N -Carbamylase– d -Hydantoinase Fusion Enzyme

Kim

Lee

Kim

2000

Appl Environ Microbiol

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gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.In the field of molecular biology and biotechnology, enzymes possessing two or more combined activities, along with appropriate stability, have found wide application (25). Although the natural diversity of the enzymes provides some candidates that have evolved to possess bifunctional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes based on evolutionary traits and well-defined structure (1, 25). Artificial fusion enzymes, simply generated by either end-to-end fusion or by tethering of whole genes encoding intact functional proteins with a linker, have been reported to show noticeable performance in a concerted fashion (6, 21).Nonnatural D-amino acids are widely used in the pharmaceutical field, with applications such as antimicrobial and antiviral agents, artificial sweeteners, pesticides, and pyrethroids (26,28,30). Due to the great commercial demand for various D-amino acids, enzymatic and chemoenzymatic routes have been developed (29). Of these, a fully enzymatic process using two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity (8). In this process, hydantoin derivative is hydrolyzed by D-hydantoinase, and the resulting N-carbamyl-D-amino acid is further converted to the corresponding D-amino acid by N-carbamylase. Therefore, functional fusion of two enzymes was expected to have several advantages over individual enzymes with respect to reaction kinetics and enzyme production, as well as novel properties and reactivity. As practical cases of the multistep sequential reaction, the performance of the hybrid or fusion enzymes sometimes was better than that achieved by successive action of individual enzymes, expanding the potential use of natural enzymes (6,21,25).Previously, we cloned and expressed two hydantoinase genes from Bacillus stearothermophilus SD1 (17) and Bacillus thermocatenulatus GH2 (15). We also identified some conserved domains possessing essential amino acid residues by comparative analyses of functionally related enzymes (16). These results provided some evidence that supports the presence of a cyclic amidohydrolase family including hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase. Further study of the deletion mutants derived from two D-hydantoinases suggested that the N terminus of D-hydantoinase is not essential for maintaining the enzyme structure and is dispensable for enzyme activity (15,17). From these observations, we made a hypothesi...

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“…More generally, there has also been considerable success in generating chimeric enzymes with novel enzymatic activities such as new substrate specificities, altered catalytic properties, and enhanced expression levels (27). Here, regulated directionality and complexity are conferred in a simple chimeric construction.…”

Section: Discussionmentioning

confidence: 99%

A chimeric Cre recombinase with regulated directionality

Warren

Laxmikanthan²,

Landy³

2008

Proc. Natl. Acad. Sci. U.S.A.

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From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long-and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.bacteriophage ͉ recombinase ͉ site-specific recombination ͉ evolution

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Hybrid enzymes: manipulating enzyme design

Cited by 81 publications

References 56 publications

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Construction and Evaluation of a Novel Bifunctional N -Carbamylase– d -Hydantoinase Fusion Enzyme

A chimeric Cre recombinase with regulated directionality

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