Small molecule inhibition of a Group II chaperonin: pinpointing a loop region within the equatorial domain as necessary for protein refolding.

The functionality of regions within the equatorial domain of Group II chaperonins is poorly understood. Previously we showed that a 70 amino acid sequence within this domain on the single-subunit recombinant thermosome from Methanocaldococcus jannaschii (rTHS) contains residues directly responsible for refolding protein substrates [L.M. Bergeron, C. Lee, D.S. Clark, Identification of a critical chaperoning region on an archaeal recombinant thermosome, Biochem. Biophys. Res. Commun. 369 (2008) 707-711]. In the present study, 6-aminopenicillanic acid (6-APA) was found to bind to rTHS and inhibit it from refolding proteins. Fluorescence anisotropy was used to measure a 6-APA/rTHS dissociation constant of 17.1 microM and verify that the binding site is within the first 70 amino-terminal rTHS residues. Docking simulations point to a specific loop region at residues 53-57 on rTHS as the most likely binding region. This loop region is located within the oligomeric association sites of the wild-type thermosome. These results implicate a specific equatorial region of Group II chaperonins in the refolding of proteins, and suggest its importance in conformational changes that accompany chaperone function.

Redirecting the inactivation pathway of penicillin amidase and increasing amoxicillin production via a thermophilic molecular chaperone.

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.

Self-renaturing enzymes: design of an enzyme-chaperone chimera as a new approach to enzyme stabilization.

Molecular chaperones in aqueous-organic mixtures can broaden the utility of biocatalysis by stabilizing enzymes in denaturing conditions. We have designed a self-renaturing enzyme-chaperone chimera consisting of penicillin amidase and a thermophilic chaperonin that functions in aqueous-organic mixtures. The flexible linker separating the enzyme and chaperone domains was optimized and the design was extended to incorporate a chitin binding domain to facilitate immobilization of the chimera to a chitin support. The initial specific activity of penicillin amidase was not compromised by the enzyme-chaperone fusion or by immobilization. The total turnover number of immobilized chimera for amoxicillin synthesis in aqueous-methanol mixtures was 2.8 times higher after 95 h than the total turnover number of the immobilized penicillin amidase lacking a chaperone domain. Similarly, in 32% methanol the soluble chimera was active for over three times longer than the enzyme alone. This approach could easily be extended to other enzyme systems.