8EKG

MHETase variant Thr159Val, Met192Tyr, Tyr252Phe, Tyr503Trp

Summary for 8EKG

• Entry DOI: 10.2210/pdb8ekg/pdb
• Descriptor: Mono(2-hydroxyethyl) terephthalate hydrolase, CALCIUM ION, DI(HYDROXYETHYL)ETHER, ... (6 entities in total)
• Functional Keywords: protein engineering, mhetase, plastic degradation, hydrolase
• Biological source: Ideonella sakaiensis
• Total number of polymer chains: 6
• Total formula weight: 375648.07

Authors

Saunders, J.W.,Frkic, R.L.,Jackson, C.J. (deposition date: 2022-09-20, release date: 2023-10-18, Last modification date: 2024-11-06)

Primary citation

Saunders, J.W.,Damry, A.M.,Vongsouthi, V.,Spence, M.A.,Frkic, R.L.,Gomez, C.,Yates, P.A.,Matthews, D.S.,Tokuriki, N.,McLeod, M.D.,Jackson, C.J.
Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.
Biochemistry, 63:1663-1673, 2024

PubMed Abstract: The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

PubMed: 38885634
DOI: 10.1021/acs.biochem.4c00165

Experimental method

X-RAY DIFFRACTION (2.65 Å)