4KQX

Mutant Slackia exigua KARI DDV in complex with NAD and an inhibitor

PDB DOI: https://doi.org/10.2210/pdb4KQX/pdb

Classification: OXIDOREDUCTASE/OXIDOREDUCTASE INHIBITOR
Organism(s): Slackia exigua ATCC 700122
Expression System: Escherichia coli BL21(DE3)
Mutation(s): Yes

Deposited: 2013-05-15 Released: 2013-06-26
Deposition Author(s): Brinkmann-Chen, S., Flock, T., Cahn, J.K.B., Snow, C.D., Brustad, E.M., Mcintosh, J.A., Meinhold, P., Zhang, L., Arnold, F.H.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.80 Å
R-Value Free: 0.252
R-Value Work: 0.201
R-Value Observed: 0.204

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.2 of the entry. See complete history.

Literature

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

Brinkmann-Chen, S., Flock, T., Cahn, J.K., Snow, C.D., Brustad, E.M., McIntosh, J.A., Meinhold, P., Zhang, L., Arnold, F.H.

(2013) Proc Natl Acad Sci U S A 110: 10946-10951

PubMed: 23776225 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1073/pnas.1306073110
Primary Citation of Related Structures:
4KQW, 4KQX

PubMed Abstract:
To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch.

Organizational Affiliation

California Institute of Technology, Pasadena, CA 91125, USA.

Macromolecule Content

Total Structure Weight: 79.88 kDa
Atom Count: 5,497
Modelled Residue Count: 657
Deposited Residue Count: 700
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Ketol-acid reductoisomerase	A, B	350	Slackia exigua ATCC 700122	Mutation(s): 3 Gene Names: ilvC, HMPREF0762_00954 EC: 1.1.1.86
UniProt
Find proteins for D0WGK0 (Slackia exigua (strain ATCC 700122 / DSM 15923 / CIP 105133 / JCM 11022 / KCTC 5966 / S-7)) Explore D0WGK0 Go to UniProtKB: D0WGK0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	D0WGK0
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 4 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
NAD Query on NAD Download Ideal Coordinates CCD File SDF format, chain C [auth A] SDF format, chain J [auth B] MOL2 format, chain C [auth A] MOL2 format, chain J [auth B] mmCIF format, chain C [auth A] mmCIF format, chain J [auth B]	C [auth A], J [auth B]	NICOTINAMIDE-ADENINE-DINUCLEOTIDE C₂₁ H₂₇ N₇ O₁₄ P₂ BAWFJGJZGIEFAR-NNYOXOHSSA-N		Interactions Focus chain C [auth A] Focus chain J [auth B] Interactions & Density Focus chain C [auth A] Focus chain J [auth B]
HIS Query on HIS Download Ideal Coordinates CCD File SDF format, chain K [auth B] MOL2 format, chain K [auth B] mmCIF format, chain K [auth B]	K [auth B]	HISTIDINE C₆ H₁₀ N₃ O₂ HNDVDQJCIGZPNO-YFKPBYRVSA-O		Interactions Focus chain K [auth B] Interactions & Density Focus chain K [auth B]
HIO Query on HIO Download Ideal Coordinates CCD File SDF format, chain D [auth A] SDF format, chain I [auth B] MOL2 format, chain D [auth A] MOL2 format, chain I [auth B] mmCIF format, chain D [auth A] mmCIF format, chain I [auth B]	D [auth A], I [auth B]	N-HYDROXY-N-ISOPROPYLOXAMIC ACID C₅ H₉ N O₄ QVIOSGUKMDGWNN-UHFFFAOYSA-N		Interactions Focus chain D [auth A] Focus chain I [auth B] Interactions & Density Focus chain D [auth A] Focus chain I [auth B]
MG Query on MG Download Ideal Coordinates CCD File SDF format, chain E [auth A] SDF format, chain F [auth A] SDF format, chain G [auth A] SDF format, chain H [auth A] SDF format, chain L [auth B] 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] MOL2 format, chain E [auth A] MOL2 format, chain F [auth A] MOL2 format, chain G [auth A] MOL2 format, chain H [auth A] MOL2 format, chain L [auth B] 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] mmCIF format, chain E [auth A] mmCIF format, chain F [auth A] mmCIF format, chain G [auth A] mmCIF format, chain H [auth A] mmCIF format, chain L [auth B] 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]	E [auth A] F [auth A] G [auth A] H [auth A] L [auth B] E [auth A], F [auth A], G [auth A], H [auth A], L [auth B], M [auth B], N [auth B], O [auth B], P [auth B], Q [auth B]	MAGNESIUM ION Mg JLVVSXFLKOJNIY-UHFFFAOYSA-N		Interactions Focus chain E [auth A] Focus chain F [auth A] Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth B] 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] Interactions & Density Focus chain E [auth A] Focus chain F [auth A] Focus chain G [auth A] Focus chain H [auth A] Focus chain L [auth B] 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]

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 1.80 Å
R-Value Free: 0.252
R-Value Work: 0.201
R-Value Observed: 0.204
Space Group: P 2₁ 2₁ 2₁

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 49.985	α = 90
b = 105.346	β = 90
c = 122.357	γ = 90

Software Package:

Software Name	Purpose
Blu-Ice	data collection
PHASER	phasing
REFMAC	refinement
XSCALE	data scaling
SCALA	data scaling

Structure Validation

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Ligand Structure Quality Assessment

Entry History

Deposition Data

Released Date: 2013-06-26

Deposition Author(s):

Revision History (Full details and data files)

Version 1.0: 2013-06-26
Type: Initial release
Version 1.1: 2013-07-17
Changes: Database references
Version 1.2: 2023-09-20
Changes: Data collection, Database references, Derived calculations, Refinement description

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Help and Resources

4KQX

Mutant Slackia exigua KARI DDV in complex with NAD and an inhibitor

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

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)