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Hybrid Enzyme (hybrid + enzyme)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

An active triple-catalytic hybrid enzyme engineered by linking cyclo-oxygenase isoform-1 to prostacyclin synthase that can constantly biosynthesize prostacyclin, the vascular protector

FEBS JOURNAL, Issue 23 2008
Ke-He Ruan
It remains a challenge to achieve the stable and long-term expression (in human cell lines) of a previously engineered hybrid enzyme [triple-catalytic (Trip-cat) enzyme-2; Ruan KH, Deng H & So SP (2006) Biochemistry45, 14003,14011], which links cyclo-oxygenase isoform-2 (COX-2) to prostacyclin (PGI2) synthase (PGIS) for the direct conversion of arachidonic acid into PGI2 through the enzyme's Trip-cat functions. The stable upregulation of the biosynthesis of the vascular protector, PGI2, in cells is an ideal model for the prevention and treatment of thromboxane A2 (TXA2)-mediated thrombosis and vasoconstriction, both of which cause stroke, myocardial infarction, and hypertension. Here, we report another case of engineering of the Trip-cat enzyme, in which human cyclo-oxygenase isoform-1, which has a different C-terminal sequence from COX-2, was linked to PGI2 synthase and called Trip-cat enzyme-1. Transient expression of recombinant Trip-cat enzyme-1 in HEK293 cells led to 3,5-fold higher expression capacity and better PGI2 -synthesizing activity as compared to that of the previously engineered Trip-cat enzyme-2. Furthermore, an HEK293 cell line that can stably express the active new Trip-cat enzyme-1 and constantly synthesize the bioactive PGI2 was established by a screening approach. In addition, the stable HEK293 cell line, with constant production of PGI2, revealed strong antiplatelet aggregation properties through its unique dual functions (increasing PGI2 production while decreasing TXA2 production) in TXA2 synthase-rich plasma. This study has optimized engineering of the active Trip-cat enzyme, allowing it to become the first to stably upregulate PGI2 biosynthesis in a human cell line, which provides a basis for developing a PGI2 -producing therapeutic cell line for use against vascular diseases. [source]

Hybrid reuteransucrase enzymes reveal regions important for glucosidic linkage specificity and the transglucosylation/hydrolysis ratio

FEBS JOURNAL, Issue 23 2008
Slavko Kralj
The reuteransucrase enzymes of Lactobacillus reuteri strain 121 (GTFA) and L. reuteri strain ATCC 55730 (GTFO) convert sucrose into ,- d -glucans (labelled reuterans) with mainly ,-(1,4) glucosidic linkages (50% and 70%, respectively), plus ,-(1,6) linkages. In the present study, we report a detailed analysis of various hybrid GTFA/O enzymes, resulting in the identification of specific regions in the N-termini of the catalytic domains of these proteins as the main determinants of glucosidic linkage specificity. These regions were divided into three equal parts (A1,3; O1,3), and used to construct six additional GTFA/O hybrids. All hybrid enzymes were able to synthesize ,-glucans from sucrose, and oligosaccharides from sucrose plus maltose or isomaltose as acceptor substrates. Interestingly, not only the A2/O2 regions, with the three catalytic residues, affect glucosidic linkage specificity, but also the upstream A1/O1 regions make a strong contribution. Some GTFO derived hybrid/mutant enzymes displayed strongly increased transglucosylation/hydrolysis activity ratios. The reduced sucrose hydrolysis allowed the much improved conversion of sucrose into oligo- and polysaccharide products. Thus, the glucosidic linkage specificity and transglucosylation/hydrolysis ratios of reuteransucrase enzymes can be manipulated in a relatively simple manner. This engineering approach has yielded clear changes in oligosaccharide product profiles, as well as a range of novel reuteran products differing in ,-(1,4) and ,-(1,6) linkage ratios. [source]

Active TEM-1 ,-lactamase mutants with random peptides inserted in three contiguous surface loops

PROTEIN SCIENCE, Issue 10 2006
Pascale Mathonet
Abstract Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non-natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM-1 ,-lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two ,-strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well-conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N-terminal helix and the first ,-strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final ,-strand to the C-terminal helix, particularly if the wild-type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium-sized loops. The combination of these activity-selected libraries generated a huge library containing active hybrid enzymes with all three loops modified. [source]