8EKG

MHETase variant Thr159Val, Met192Tyr, Tyr252Phe, Tyr503Trp

PDB DOI: https://doi.org/10.2210/pdb8EKG/pdb

Classification: HYDROLASE
Organism(s): Piscinibacter sakaiensis
Expression System: Escherichia coli
Mutation(s): Yes

Deposited: 2022-09-20 Released: 2023-10-18
Deposition Author(s): Saunders, J.W., Frkic, R.L., Jackson, C.J.
Funding Organization(s): Not funded

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.65 Å
R-Value Free: 0.241
R-Value Work: 0.184
R-Value Observed: 0.187

wwPDB Validation 3D Report Full Report

This is version 1.3 of the entry. See complete history.

Literature

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

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.

(2024) Biochemistry 63: 1663-1673

PubMed: 38885634 Search on PubMed
DOI: https://doi.org/10.1021/acs.biochem.4c00165
Primary Citation of Related Structures:
8EKG

PubMed Abstract:
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis 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.

Organizational Affiliation

Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.

Macromolecule Content

Total Structure Weight: 375.65 kDa
Atom Count: 25,228
Modelled Residue Count: 3,344
Deposited Residue Count: 3,552
Unique protein chains: 1

Macromolecules

Find similar proteins by:

(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Mono(2-hydroxyethyl) terephthalate hydrolase	A, B, C, D, E A, B, C, D, E, F	592	Piscinibacter sakaiensis	Mutation(s): 4 Gene Names: ISF6_0224 EC: 3.1.1.102
UniProt
Find proteins for A0A0K8P8E7 (Piscinibacter sakaiensis) Explore A0A0K8P8E7 Go to UniProtKB: A0A0K8P8E7
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	A0A0K8P8E7
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 4 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
PEG Query on PEG Download Ideal Coordinates CCD File SDF format, chain H [auth A] MOL2 format, chain H [auth A] mmCIF format, chain H [auth A]	H [auth A]	DI(HYDROXYETHYL)ETHER C₄ H₁₀ O₃ MTHSVFCYNBDYFN-UHFFFAOYSA-N		Interactions Focus chain H [auth A] Interactions & Density Focus chain H [auth A]
SO4 Query on SO4 Download Ideal Coordinates CCD File SDF format, chain K [auth B] SDF format, chain L [auth B] SDF format, chain Y [auth E] SDF format, chain Z [auth E] MOL2 format, chain K [auth B] MOL2 format, chain L [auth B] MOL2 format, chain Y [auth E] MOL2 format, chain Z [auth E] mmCIF format, chain K [auth B] mmCIF format, chain L [auth B] mmCIF format, chain Y [auth E] mmCIF format, chain Z [auth E]	K [auth B], L [auth B], Y [auth E], Z [auth E]	SULFATE ION O₄ S QAOWNCQODCNURD-UHFFFAOYSA-L		Interactions Focus chain K [auth B] Focus chain L [auth B] Focus chain Y [auth E] Focus chain Z [auth E] Interactions & Density Focus chain K [auth B] Focus chain L [auth B] Focus chain Y [auth E] Focus chain Z [auth E]
EDO Query on EDO Download Ideal Coordinates CCD File SDF format, chain AA [auth E] SDF format, chain I [auth A] SDF format, chain M [auth B] SDF format, chain N [auth B] SDF format, chain O [auth B] SDF format, chain P [auth B] SDF format, chain Q [auth B] SDF format, chain R [auth B] SDF format, chain T [auth C] SDF format, chain V [auth D] SDF format, chain W [auth D] MOL2 format, chain AA [auth E] MOL2 format, chain I [auth A] MOL2 format, chain M [auth B] MOL2 format, chain N [auth B] MOL2 format, chain O [auth B] MOL2 format, chain P [auth B] MOL2 format, chain Q [auth B] MOL2 format, chain R [auth B] MOL2 format, chain T [auth C] MOL2 format, chain V [auth D] MOL2 format, chain W [auth D] mmCIF format, chain AA [auth E] mmCIF format, chain I [auth A] mmCIF format, chain M [auth B] mmCIF format, chain N [auth B] mmCIF format, chain O [auth B] mmCIF format, chain P [auth B] mmCIF format, chain Q [auth B] mmCIF format, chain R [auth B] mmCIF format, chain T [auth C] mmCIF format, chain V [auth D] mmCIF format, chain W [auth D]	AA [auth E] I [auth A] M [auth B] N [auth B] O [auth B] AA [auth E], I [auth A], M [auth B], N [auth B], O [auth B], P [auth B], Q [auth B], R [auth B], T [auth C], V [auth D], W [auth D]	1,2-ETHANEDIOL C₂ H₆ O₂ LYCAIKOWRPUZTN-UHFFFAOYSA-N		Interactions Focus chain AA [auth E] Focus chain I [auth A] Focus chain M [auth B] Focus chain N [auth B] Focus chain O [auth B] Focus chain P [auth B] Focus chain Q [auth B] Focus chain R [auth B] Focus chain T [auth C] Focus chain V [auth D] Focus chain W [auth D] Interactions & Density Focus chain AA [auth E] Focus chain I [auth A] Focus chain M [auth B] Focus chain N [auth B] Focus chain O [auth B] Focus chain P [auth B] Focus chain Q [auth B] Focus chain R [auth B] Focus chain T [auth C] Focus chain V [auth D] Focus chain W [auth D]
CA Query on CA Download Ideal Coordinates CCD File SDF format, chain BA [auth F] SDF format, chain G [auth A] SDF format, chain J [auth B] SDF format, chain S [auth C] SDF format, chain U [auth D] SDF format, chain X [auth E] MOL2 format, chain BA [auth F] MOL2 format, chain G [auth A] MOL2 format, chain J [auth B] MOL2 format, chain S [auth C] MOL2 format, chain U [auth D] MOL2 format, chain X [auth E] mmCIF format, chain BA [auth F] mmCIF format, chain G [auth A] mmCIF format, chain J [auth B] mmCIF format, chain S [auth C] mmCIF format, chain U [auth D] mmCIF format, chain X [auth E]	BA [auth F] G [auth A] J [auth B] S [auth C] U [auth D] BA [auth F], G [auth A], J [auth B], S [auth C], U [auth D], X [auth E]	CALCIUM ION Ca BHPQYMZQTOCNFJ-UHFFFAOYSA-N		Interactions Focus chain BA [auth F] Focus chain G [auth A] Focus chain J [auth B] Focus chain S [auth C] Focus chain U [auth D] Focus chain X [auth E] Interactions & Density Focus chain BA [auth F] Focus chain G [auth A] Focus chain J [auth B] Focus chain S [auth C] Focus chain U [auth D] Focus chain X [auth E]