Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Enzyme-catalyzed synthesis of carbohydrates.

Several new enzymes of utility in the synthesis of carbohydrates have been reported during the past year. Additionally, the utility of several well studied enzymes has been expanded. Pyruvate aldolases, aldolase abzymes and both wild-type and mutated glycosidases have found increasing acceptance in the community. Preliminary reports suggest that thermophilic enzymes may possess significant advantages compared to their mesophilic counterparts for carbohydrate synthesis.

Cascade liposomal triggering: light-induced Ca2+ release from diplasmenylcholine liposomes triggers PLA2-catalyzed hydrolysis and contents leakage from DPPC liposomes.

We have previously reported a direct triggering approach [Thompson, D. H., et al. (1996) Biochim. Biophys. Acta 1279, 25-34; Gerasimov, O. V., et al. (1997) Biochim. Biophys. Acta 1324, 200-214] based on the facile degradation of plasmenylcholine and diplasmenylcholine vinyl ether linkages by either photooxidation or low-pH environments. This report describes a novel, cascade-type triggering technique that utilizes liposome photooxidation and contents release to activate an enzyme capable of destabilizing conventional phosphatidylcholine liposomes. Our application of this concept employs a mixture of two different liposome populations, one composed of synthetic diplasmenylcholine (1, 2-dihexadec-1'-enyl-sn-glycero-3-phosphocholine, DPPlsCho) containing Ca2+ as a signaling agent for phospholipase A2 (PLA2) and the second composed of 1, 2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) with encapsulated calcein as the reporter molecule. Bacteriochlorophyll (BChl)-sensitized photorelease of Ca2+ from PLA2-resistant DPPlsCho liposomes activates extravesicular PLA2, thereby promoting catalyzed DPPC hydrolysis in a secondary triggering reaction, leading to calcein release. BChl/DPPlsCho/DHC/DPPE-PEG5000/Ca2+IN (0.5:85:10:5) liposomes can be phototriggered using 800 nm excitation, resulting in Ca2+ release (t50% release = 15 min) that cocatalyzes the release of calcein (t50% release = 40 min) from DPPC liposomes (1.5 mM total lipid in DPPlsCho liposomes, 0.18 mM DPPC, 210 micro M final Ca2+ concentration, 90 units of PLA2/ml, 50 mM calcein, and 36 micro M EDTA). No appreciable calcein release occurs in the absence of either PLA2 or BChl/DPPlsCho/DHC/DPPE-PEG5000/CaIN liposomes. The implications of this cascade triggering technique on drug delivery approaches are briefly discussed.