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Hybrid enzymes
Hybrid enzymes are produced through genetic engineering, each enzyme containing elements of two or more enzymes, thus expanding the potential uses of natural enzymes. These hybrid enzymes are becoming important source of novel enzymes. Although they are believed to have been generated in nature during evolution, they can also be produced in the laboratory in a number of ways including the following: (i) a single point mutation or a secondary structure (e.g., an α-helix) may be substituted from one enzyme into another enzyme; (H) a secondary-structural element (e.g. an α-helix), a whole domain or a monomeric unit of a multimeric enzyme may be transferred or exchanged from another enzyme, or fusions between two enzymes having separate and distinct activities may be brought about artificially. The alterations in enzymes through production of hybrid enzymes involve both non-catalytic and catalytic properties.
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| Generation of Hybrid Enzymes by Substitution of Point Mutation/Secondary Structures From Enzyme A into Enzyme B |
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| Generation of hybrid enzymes by substitution of point mutation/ secomdary structures from enzyme A inot Enzyme B |
| 1. Helix transferred |
2. Helix transferred |
3. Point mutations |
4. Point mutations |
| 5. Secondary structure |
6. Point mutations and secondary strucutre |
7. Point mutations |
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Alteration in non-catalytic properties
Non-enzymatic properties of an enzyme include its thermostability and the activity level. This will be illustrated using three examples. (i) Hybrids of β-g1ucosidases from Agrobacterium tumefaciens (pH 7.2-7.4; 60°C) and Cellvibrio gilvus (pH 6.2-6.4; 350°C) had optimum activity at intermediate pH (6.6-7.0) and temperature (45-50°C) and also had KM value intermediate between the two parental enzymes. (H) The activity level of β-lactamase has been altered by producing hybrid enzymes.
For instance, β-lactamases from RTEM-l and Proteus vulgaris having 37% similarity were used for producing hybrid enzymes; although many hybrids had no activity, but a few had activity, which could be successfully improved through mutagenesis. (Hi) Insertion of a short loop of defined function may also lead to altered activity. For instance, interleukin-1β (IL-1β) is insensitive to proteases, but insertion of a protease binding sequence in this enzyme, created an elastase and chymotrypsin-sensitive IL-1β.
Creation of enzymes with novel activities.
Hybrid enzymes may also be produced, which alter their functionality. This is sometimes achieved through introduction of elements with known properties into an existing enzyme. This process is described as retrofitting.
The alteration of functionality of enzymes can be possible at three levels: (i) specificity or catalytic activity of the enzyme may be altered through mutations, (ii) catalytic residues may be introduced into a binding protein having no catalytic activity and (iii) both catalytic and binding properties may be introduced into a protein scaffold. These changes lead to alterations in the values of kinetic parameters (KM kcat kcat/KM) as illustrated by some examples listed in and described in the following para.
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(a) Modulation of specificity.
Specificity is an attribute which has been modulated in the largest number of hybrid enzymes. Even a single amino acid alteration has been shown in some cases to change specificity, which is exemplified by conversion of glutaconate CoA (from Acidaminococcus fermentans) into acyl-CoAhydrolase. In some other cases, exchanges of amino acid residues or point mutations in the coenzyme binding domains resulted in changes in their cofactor specificity preferences (e.g. preference of glutathione reductase and lipoamide dehydrogenase, from NAD to NADP or vice versa; see.
Following are some examples: (i) Exchange of some amino acid residues in coenzyme binding site of glutathione reductase and lipoamide dehydrogenase resulted in alterations in cofactor preferences from NAD to NADP and vice versa. (ii) A change in substrate specificity was also possible in lactate dehydrogenase from B. stearothermophilus. (iii) In isopropylmalate dehydrogenase from Thermus thermophilus, replacement of a β turn by a 13-residue α helix (from a NADP utilizing E. coli) and four other point mutations resulted in a shift in preference to NADP by a factor of 105. Another method of creating novel hybrid enzymes is the exchange of functional domains, described as DNA shuffing or DNA swapping.
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This approach is particularly suitable for modular enzymes. Following are some examples: (i) In E. coli, glycinamide-ribonucleotide (GAR) transformylase (called PurN) and N10-formyltetrahydrofolate hydrolase (called PurU) utilize the same cofactor N10-formyltetrahydrofolate. PurN has two domains (a ribonucleotide binding domain and the other a cofactor-binding domain containing catalytic site for formyl-group transfer), of which the cofactor-catalytic domain seems to be homologous to that in PurU.
Several PurN-PurU hybrid enzymes, carrying GAR binding domain from PurN and cofactor catalytic domain from PurU were designed using DNA swapping approach, and one of them complemented an E. coli mutant, deficient for GAR-transformylase activity.
Restriction enzymes with novel specificities were also produced using the above approach of DNA swapping. For instance, a hybrid enzyme has been created by fusing the cleavage domain of FokI with DNA binding motif from Ubx homeodomain of Drosophila and with consensus zinc-finger proteins (zinc-fingers can be engineered for binding to any DNA sequence).
Hybrid enzymes for synthesis of secondary metabolites (e.g. small peptides, polyketides and terpenes) have also been produced, due to modular nature of these enzymes. One such enzyme is SrfA complex (Bacillus subtilis), which generated peptides for the synthesis of a lipopeptide, an antibiotic or a surfactin.
The gene srfA has a Leu-activating domain, which can be replaced by Phe-, Om amd Valdomains from Penicillium chrysogenum. These hybrid genes encoded peptide synthetases with the desired amino acid specificities and produced peptides with corresponding amino acid substitutions.
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Hybrid enzymes (e.g., multifunctional polyketide- synthase) are also used for the synthesis of polyketides, a groups of secondary metabolites obtained through repeated condensation of acetyl and malonyl units to produce compounds like antibiotics, anticancer agents or immuno- suppressants. Exchange of catalytic units is achieved through 'mix and match' strategy to produce hybrid polyketide synthase enzymes.
For instance, a 6-deoxyerythronolide-B synthase (DEBS) from an yeast had its ketoreductase domain replaced by domains from rapamycin polyketide synthase (RAPs), to produce two hybrid DEBS enzymes (a decarboxylated tetraketide and an eight membered-ring tetraketide lactone). Similar other hybrid polyketide synthasesa were porduced, which had altered specificity. These examples illustrate the feasibility of the in vivo generation of specific secondary metabolites by domain swapping.
(b) Introduction of catalytic residues into a binding protein.
There seems to be no example (atleast not till 1998), where catalytic activity was generated through introduction of catalytic residues into a binding protein, but theoretical possibilities exist, where a glucose-binding protein may be converted into a hexokinase and an antibody is converted into an enzyme (catalytic antibody).
(c) Introduction of residues for both catalysis and binding
In this approach, one s arts with a suitable scaffold, and introduces an active site that imparts both, the substrate specificity and the desired catalytic properties. An algorithm DEZYMER is also available to search 'through protein structures to identify regions having the desired active-site geometry. It was used to introduce a nonheme-iron active site into thioredoxin having no iron-binding site in nature. The resulting enzyme was capable of superoxide dismutation at a rate 105M-1S-1 as against the natural rate or109M-1S-1.
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