4PGL

Crystal structure of engineered D-tagatose 3-epimerase PcDTE-ILS6

PDB DOI: https://doi.org/10.2210/pdb4PGL/pdb

Classification: ISOMERASE
Organism(s): Pseudomonas cichorii
Expression System: Escherichia coli BL21(DE3)
Mutation(s): Yes

Deposited: 2014-05-02 Released: 2014-10-22
Deposition Author(s): Hee, C.S., Bosshart, A., Schirmer, T.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.10 ?
R-Value Free: 0.181
R-Value Work: 0.152

Starting Model: experimental
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Ligand Structure Quality Assessment

This is version 1.5 of the entry. See complete history.

Literature

Directed Divergent Evolution of a Thermostable D-Tagatose Epimerase towards Improved Activity for Two Hexose Substrates.

Bosshart, A., Hee, C.S., Bechtold, M., Schirmer, T., Panke, S.

(2015) Chembiochem 16: 592-601

PubMed: 25655925 Search on PubMed
DOI: https://doi.org/10.1002/cbic.201402620
Primary Citation of Related Structures:
4PFH, 4PGL, 4Q7I

PubMed Abstract:
Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D-tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shells around the substrate-binding site of Var8 yielded the eight-site mutant IDF8 (ninefold improved kcat for the epimerization of D-fructose) and the six-site mutant ILS6 (14-fold improved epimerization of L-sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase.

Organizational Affiliation

Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058 Basel (Switzerland).

Macromolecule Content

Total Structure Weight: 137.93 kDa
Atom Count: 9,924
Modelled Residue Count: 1,176
Deposited Residue Count: 1,192
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
D-tagatose 3-epimerase	A, B, C, D	298	Pseudomonas cichorii	Mutation(s): 14 EC: 5.3.1 (PDB Primary Data), 5.1.3.31 (UniProt)
UniProt
Find proteins for O50580 (Pseudomonas cichorii) Explore O50580 Go to UniProtKB: O50580
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	O50580
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 4 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
LTG Query on LTG Download Ideal Coordinates CCD File SDF format, chain I [auth A] SDF format, chain M [auth B] SDF format, chain Q [auth C] SDF format, chain U [auth D] MOL2 format, chain I [auth A] MOL2 format, chain M [auth B] MOL2 format, chain Q [auth C] MOL2 format, chain U [auth D] mmCIF format, chain I [auth A] mmCIF format, chain M [auth B] mmCIF format, chain Q [auth C] mmCIF format, chain U [auth D]	I [auth A], M [auth B], Q [auth C], U [auth D]	L-tagatose C₆ H₁₂ O₆ BJHIKXHVCXFQLS-LFRDXLMFSA-N		Interactions Focus chain I [auth A] Focus chain M [auth B] Focus chain Q [auth C] Focus chain U [auth D] Interactions & Density Focus chain I [auth A] Focus chain M [auth B] Focus chain Q [auth C] Focus chain U [auth D]
SOE Query on SOE Download Ideal Coordinates CCD File SDF format, chain G [auth A] SDF format, chain H [auth A] SDF format, chain L [auth B] SDF format, chain P [auth C] SDF format, chain T [auth D] MOL2 format, chain G [auth A] MOL2 format, chain H [auth A] MOL2 format, chain L [auth B] MOL2 format, chain P [auth C] MOL2 format, chain T [auth D] mmCIF format, chain G [auth A] mmCIF format, chain H [auth A] mmCIF format, chain L [auth B] mmCIF format, chain P [auth C] mmCIF format, chain T [auth D]	G [auth A], H [auth A], L [auth B], P [auth C], T [auth D]	alpha-L-sorbopyranose C₆ H₁₂ O₆ LKDRXBCSQODPBY-BGPJRJDNSA-N		Interactions Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth B] Focus chain P [auth C] Focus chain T [auth D] Interactions & Density Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth B] Focus chain P [auth C] Focus chain T [auth D]
SOL Query on SOL Download Ideal Coordinates CCD File SDF format, chain F [auth A] SDF format, chain N [auth B] SDF format, chain R [auth C] SDF format, chain V [auth D] MOL2 format, chain F [auth A] MOL2 format, chain N [auth B] MOL2 format, chain R [auth C] MOL2 format, chain V [auth D] mmCIF format, chain F [auth A] mmCIF format, chain N [auth B] mmCIF format, chain R [auth C] mmCIF format, chain V [auth D]	F [auth A], N [auth B], R [auth C], V [auth D]	L-sorbose C₆ H₁₂ O₆ BJHIKXHVCXFQLS-OTWZMJIISA-N		Interactions Focus chain F [auth A] Focus chain N [auth B] Focus chain R [auth C] Focus chain V [auth D] Interactions & Density Focus chain F [auth A] Focus chain N [auth B] Focus chain R [auth C] Focus chain V [auth D]
MN Query on MN Download Ideal Coordinates CCD File SDF format, chain E [auth A] SDF format, chain J [auth A] SDF format, chain K [auth B] SDF format, chain O [auth C] SDF format, chain S [auth D] MOL2 format, chain E [auth A] MOL2 format, chain J [auth A] MOL2 format, chain K [auth B] MOL2 format, chain O [auth C] MOL2 format, chain S [auth D] mmCIF format, chain E [auth A] mmCIF format, chain J [auth A] mmCIF format, chain K [auth B] mmCIF format, chain O [auth C] mmCIF format, chain S [auth D]	E [auth A], J [auth A], K [auth B], O [auth C], S [auth D]	MANGANESE (II) ION Mn WAEMQWOKJMHJLA-UHFFFAOYSA-N		Interactions Focus chain E [auth A] Focus chain J [auth A] Focus chain K [auth B] Focus chain O [auth C] Focus chain S [auth D] Interactions & Density Focus chain E [auth A] Focus chain J [auth A] Focus chain K [auth B] Focus chain O [auth C] Focus chain S [auth D]

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 2.10 ?
R-Value Free: 0.181
R-Value Work: 0.152
Space Group: P 1 2₁ 1

Unit Cell:

Length ( ? )	Angle ( ? )
a = 102.81	α = 90
b = 47.44	β = 102.49
c = 126.38	γ = 90

Software Package:

Software Name	Purpose
Coot	model building
MOSFLM	data reduction
Aimless	data scaling
MOLREP	phasing

Structure Validation

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Ligand Structure Quality Assessment

Entry History

Deposition Data

Released Date: 2014-10-22

Deposition Author(s):

Hee, C.S.

Bosshart, A.

Schirmer, T.

Revision History (Full details and data files)

Version 1.0: 2014-10-22
Type: Initial release
Version 1.1: 2015-02-18
Changes: Database references
Version 1.2: 2015-03-04
Changes: Database references
Version 1.3: 2017-11-22
Changes: Derived calculations, Other, Refinement description, Source and taxonomy, Structure summary
Version 1.4: 2020-07-29
Type: Remediation
Reason: Carbohydrate remediation
Changes: Data collection, Derived calculations, Refinement description
Version 1.5: 2023-09-27
Changes: Data collection, Database references, Refinement description, Structure summary

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4PGL

Crystal structure of engineered D-tagatose 3-epimerase PcDTE-ILS6

Directed Divergent Evolution of a Thermostable D-Tagatose Epimerase towards Improved Activity for Two Hexose Substrates.

Entity ID: 1

UniProt

Entity Groups

Sequence Annotations

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Experimental Data

Structure Validation

Ligand Structure Quality Assessment

Deposition Data

Revision History (Full details and data files)