8UFY

High Resolution structure of A. viridans Lactate Oxidase

PDB DOI: https://doi.org/10.2210/pdb8UFY/pdb

Classification: FLAVOPROTEIN
Organism(s): Aerococcus viridans
Expression System: Escherichia coli BL21(DE3)
Mutation(s): No

Deposited: 2023-10-05 Released: 2024-08-14
Deposition Author(s): Lodowski, D.T.
Funding Organization(s): Other government

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 1.20 ?
R-Value Free: 0.162
R-Value Work: 0.146
R-Value Observed: 0.147

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.1 of the entry. See complete history.

Literature

An engineered lactate oxidase based electrochemical sensor for continuous detection of biomarker lactic acid in human sweat and serum.

He, Q., Wang, C., Jain, R., Byrnes, J., Farquhar, E.R., Reed, E., Berezovsky, E., Chance, M.R., Lodowski, D., An, R.

(2024) Heliyon 10: e34301-e34301

PubMed: 39149041 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1016/j.heliyon.2024.e34301
Primary Citation of Related Structures:
8UFY

PubMed Abstract:
Lactate levels in humans reveal intensity and duration of exertion and provide a critical readout for the severity of life-threatening illnesses such as pediatric sepsis. Using the lactate oxidase enzyme (Lox) from Aerococcus viridians , we demonstrated its functionality for lactate electrochemical sensing in physiological fluids in a lab setting. The structure and dynamics of LOx were validated by crystallography, X-ray scattering, and hydroxyl radical protein footprinting. This provided a validated protein template for understanding and designing an enzyme-based electrochemical sensing elements. Using this template, LOx enzyme variants were generated and compared. Comparison of the variants demonstrates that one exhibits effective lactate sensing at significantly reduced operating voltages. Additionally, we demonstrate that the four hexahistidine-tags on each enzyme tetramer are sufficient for immobilization to create a durable, functional sensor, with no need for a covalent attachment, enabling self-immobilization and eliminating the need for additional immobilization steps. The functionality of the LOx enzyme variants was verified at physiological lactate concentrations in both human serum (0-4?mM) and artificial sweat (0-100?mM) using 3-electrode setups for analysis of the three variants in parallel. Accuracy of measurement in both artificial sweat and human serum were high. Employing a microfluidic flow cell, we successfully monitored varying lactate levels in physiological fluids continuously over a 2h period. Overall, this optimized LOx enzyme, which self-immobilizes onto gold sensing electrodes, facilitates efficient and reliable lactate detection and continuous monitoring at reduced operating voltages suitable for further development towards commercial use.

Organizational Affiliation

Department of Biomedical Engineering, University of Houston, United States.

Macromolecule Content

Total Structure Weight: 84.87 kDa
Atom Count: 6,401
Modelled Residue Count: 705
Deposited Residue Count: 760
Unique protein chains: 1

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
L-lactate oxidase	A, B	380	Aerococcus viridans	Mutation(s): 0 EC: 1.1.3
UniProt
Find proteins for Q44467 (Aerococcus viridans (strain ATCC 11563 / DSM 20340 / CCUG 4311 / JCM 20461 / NBRC 12219 / NCTC 8251 / M1)) Explore Q44467 Go to UniProtKB: Q44467
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	Q44467
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 4 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
FMN (Subject of Investigation/LOI) Query on FMN Download Ideal Coordinates CCD File SDF format, chain H [auth B] SDF format, chain C [auth A] MOL2 format, chain H [auth B] MOL2 format, chain C [auth A] mmCIF format, chain H [auth B] mmCIF format, chain C [auth A]	C [auth A], H [auth B]	FLAVIN MONONUCLEOTIDE C₁₇ H₂₁ N₄ O₉ P FVTCRASFADXXNN-SCRDCRAPSA-N		Interactions Focus chain C [auth A] Focus chain H [auth B] Interactions & Density Focus chain C [auth A] Focus chain H [auth B]
LAC (Subject of Investigation/LOI) Query on LAC Download Ideal Coordinates CCD File SDF format, chain F [auth A] SDF format, chain J [auth B] MOL2 format, chain F [auth A] MOL2 format, chain J [auth B] mmCIF format, chain F [auth A] mmCIF format, chain J [auth B]	F [auth A], J [auth B]	LACTIC ACID C₃ H₆ O₃ JVTAAEKCZFNVCJ-UWTATZPHSA-N		Interactions Focus chain F [auth A] Focus chain J [auth B] Interactions & Density Focus chain F [auth A] Focus chain J [auth B]
PYR (Subject of Investigation/LOI) Query on PYR Download Ideal Coordinates CCD File SDF format, chain I [auth B] SDF format, chain G [auth A] MOL2 format, chain I [auth B] MOL2 format, chain G [auth A] mmCIF format, chain I [auth B] mmCIF format, chain G [auth A]	G [auth A], I [auth B]	PYRUVIC ACID C₃ H₄ O₃ LCTONWCANYUPML-UHFFFAOYSA-N		Interactions Focus chain G [auth A] Focus chain I [auth B] Interactions & Density Focus chain G [auth A] Focus chain I [auth B]
EDO Query on EDO Download Ideal Coordinates CCD File SDF format, chain D [auth A] SDF format, chain E [auth A] MOL2 format, chain D [auth A] MOL2 format, chain E [auth A] mmCIF format, chain D [auth A] mmCIF format, chain E [auth A]	D [auth A], E [auth A]	1,2-ETHANEDIOL C₂ H₆ O₂ LYCAIKOWRPUZTN-UHFFFAOYSA-N		Interactions Focus chain D [auth A] Focus chain E [auth A] Interactions & Density Focus chain D [auth A] Focus chain E [auth A]