L-Lactate Oxidase Activity: A New Gene Ontology Term

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Dive into the world of enzyme classification with our proposal for a new Gene Ontology (GO) term: L-lactate oxidase activity. This article details why this addition is crucial for accurate annotation and understanding of enzyme functions. Let's explore the specifics, justifications, and supporting evidence for this new term, making enzyme annotation more precise and effective.

Requested Term Details

Preferred Term Label

The preferred term label is L-lactate oxidase activity. This straightforward name clearly identifies the enzymatic function we aim to classify. Guys, it's all about making things as clear as possible, right?

Synonyms

To enhance discoverability and reflect common usage, we propose the following synonyms:

  • lactate oxidase activity
  • LOX activity
  • L-lactate:oxygen oxidoreductase activity

Definition

The definition provides a precise description of the reaction catalyzed by this enzyme:

Definition: Catalysis of the reaction: (S)-lactate + O2 = pyruvate + H2O2.

Parent Term

To maintain consistency with existing GO hierarchies, the proposed parent term is:

Parent term: GO:0003973 ((S)-2-hydroxy-acid oxidase activity)

  • This parent relationship aligns perfectly with RHEA's structure, where RHEA:55868 is a child of RHEA:16789 (a (2S)-2-hydroxycarboxylate + O2 = a 2-oxocarboxylate + H2O2), which corresponds to the general (S)-2-hydroxy-acid oxidase activity.

Ontology Branch

This term falls under the molecular_function ontology branch, as it describes a specific enzymatic activity.

Mappings

To ensure interoperability with other databases, we propose the following mappings:

  • EC Number: EC:1.1.3.2 (skos:exactMatch)
  • RHEA: RHEA:55868 (skos:exactMatch)
  • MetaCyc: LACTATE-2-MONOOXYGENASE-RXN (skos:exactMatch - note the misleading name)
  • KEGG Reaction: R00319 (skos:exactMatch)

Note on mapping consistency: The MetaCyc entry LACTATE-2-MONOOXYGENASE-RXN lists KEGG R00319 as its corresponding reaction, confirming the mapping consistency across databases despite the confusing MetaCyc identifier name.

Note to RHEA curators: The RHEA:55868 entry currently lacks a KEGG cross-reference. Consider adding R00319 as the corresponding KEGG reaction to improve database interoperability.

Justification

Addressing the Annotation Gap

Currently, the Gene Ontology lacks a specific term for L-lactate oxidase activity (EC 1.1.3.2). This omission forces curators to use the more general term GO:0003973 ((S)-2-hydroxy-acid oxidase activity), which doesn't capture the unique characteristics of this enzyme. By introducing a specific term, we can significantly improve the precision of annotations.

The absence of a specific term for L-lactate oxidase activity means that researchers and curators are left with a gap in their annotation toolkit. This gap necessitates the use of the broader term GO:0003973, which, while encompassing, fails to highlight the distinctive attributes of L-lactate oxidase. Creating a dedicated term will allow for a more granular and accurate representation of enzymatic functions, leading to better data interpretation and scientific insights. The enhanced precision will also aid in the reconstruction of metabolic pathways and the identification of specific enzymes involved in lactate metabolism, contributing to a more comprehensive understanding of cellular processes.

Biochemical Distinctiveness

L-lactate oxidase exhibits distinct biochemical characteristics that set it apart from related enzymes:

  1. Substrate specificity: Demonstrates strict specificity for (S)-lactate (L-lactate).
  2. Electron acceptor: Employs molecular oxygen (O2) as the terminal electron acceptor, producing H2O2.
  3. Cofactor requirement: Relies on an FMN-dependent oxidation mechanism.
  4. Reaction mechanism: Operates through flavin-mediated α-hydroxyacid oxidation with carbanion intermediate formation.

These characteristics distinguish it from:

  • L-lactate dehydrogenase (GO:0140171): Uses NAD+ as an electron acceptor, not O2.
  • Lactate 2-monooxygenase (GO:0050040): Produces acetate + CO2 + H2O, not pyruvate + H2O2.

The biochemical distinctiveness of L-lactate oxidase underscores the necessity for a unique GO term. Unlike L-lactate dehydrogenase, which uses NAD+ as an electron acceptor, or Lactate 2-monooxygenase, which produces acetate, L-lactate oxidase specifically uses molecular oxygen to convert L-lactate into pyruvate and H2O2. This distinction is crucial because it reflects different metabolic pathways and cellular roles. By creating a specific GO term, we acknowledge and preserve this critical biochemical information, enabling researchers to accurately identify and differentiate this enzyme from others with similar substrates but different reaction mechanisms. This clarity enhances the quality of annotations and supports more accurate interpretations of experimental data.

Historical Classification Confusion

The classification of L-lactate oxidase has a complex history, underscoring the need for clear ontological terms. The enzyme classification has faced a winding road, with periods of misclassification and outdated entries persisting in major databases. Let's break it down, guys:

Timeline of EC number changes:

  • 1961: EC 1.1.3.2 created for L-lactate oxidase
  • 1972: EC 1.1.3.2 transferred to EC 1.13.12.4, conflating two distinct activities
  • 2018: EC 1.1.3.2 reinstated as a separate enzyme class

Current status (as of 2025):

  • ExplorEnz (IUBMB): Correctly shows EC 1.1.3.2 as active for L-lactate oxidase (reaction: (S)-lactate + O2 = pyruvate + H2O2)
  • ExPASy ENZYME: Still shows EC 1.1.3.2 as "Transferred to EC 1.13.12.4" (appears to be at least 7 years out of date)
  • MetaCyc: Uses the confusing identifier "LACTATE-2-MONOOXYGENASE-RXN" for the oxidase reaction, likely a remnant from the 1972-2018 period when these were considered the same enzyme

This historical confusion arose because both enzymes:

  1. Use L-lactate as substrate
  2. Use O2 as electron acceptor
  3. Are found in similar organisms

However, they are biochemically distinct:

  • L-lactate oxidase (EC 1.1.3.2): L-lactate + O2 → pyruvate + H2O2 (simple oxidation)
  • Lactate 2-monooxygenase (EC 1.13.12.4): L-lactate + O2 → acetate + CO2 + H2O (oxidative decarboxylation)

The historical classification confusion surrounding L-lactate oxidase highlights the critical role of precise GO terms. The enzyme's journey through various EC numbers, including a period where it was conflated with Lactate 2-monooxygenase, underscores the necessity for clear and distinct ontological classifications. The persistence of outdated classifications in databases like ExPASy and the use of misleading nomenclature in MetaCyc can lead to continued misannotation and inaccurate data interpretation. By establishing a specific GO term for L-lactate oxidase, we provide a definitive and unambiguous identifier that helps prevent future errors. This clarity is crucial for maintaining data integrity and ensuring that researchers can accurately identify and annotate this enzyme, contributing to a more reliable understanding of its role in biological processes. Guys, it's like finally clearing up that one confusing family story at Thanksgiving, right?

Precedent in GO

GO already includes specific terms for many other L-amino acid and L-hydroxy acid oxidases:

  • GO:0008734 (L-aspartate oxidase activity)
  • GO:0050025 (L-glutamate oxidase activity)
  • GO:0050029 (L-lysine oxidase activity)
  • GO:0050024 (L-galactonolactone oxidase activity)
  • GO:0050035 (L-sorbose oxidase activity)

The absence of L-lactate oxidase activity is inconsistent with this established pattern, thereby proving the point of the necessity of the term.

The existence of specific GO terms for other L-amino acid and L-hydroxy acid oxidases establishes a clear precedent. Terms like L-aspartate oxidase activity (GO:0008734) and L-glutamate oxidase activity (GO:0050025) demonstrate the value of distinguishing between different oxidase activities within the GO framework. The absence of a corresponding term for L-lactate oxidase creates an inconsistency that should be rectified to maintain the logical coherence and completeness of the ontology. This addition will align the classification of L-lactate oxidase with that of other similar enzymes, ensuring that the GO accurately reflects the diversity and specificity of enzymatic functions. It's like ensuring every player on the team has their own jersey, right? Gotta keep things consistent!

Supporting Evidence

Published Literature

  • PMID:34555022 - Rembeza E, Engqvist MKM. (2021) "Experimental and computational investigation of enzyme functional annotations uncovers misannotation in the EC 1.1.3.15 enzyme class." PLoS Comput Biol.
    • Experimentally validated L-lactate oxidase activity (EC 1.1.3.2) as a distinct enzyme class
    • Demonstrated that at least 78% of sequences in related enzyme classes are misannotated
    • Highlights the need for precise functional annotation terms
  • PMID:8589073 - Lederer F. (1996) "L-lactate oxidase and L-lactate monooxygenase: mechanistic variations on a common structural theme." Biochimie.
    • Detailed mechanistic studies showing the unique catalytic mechanism of L-lactate oxidase
    • Demonstrates the flavin-mediated oxidation with H2O2 production
  • PMC:5648907 - Zaunmüller T, et al. (2017) "Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis"
    • Shows the metabolic role of L-lactate oxidase in bacterial lactate catabolism
    • Demonstrates the enzyme's role in aerobic energy metabolism

Organisms with Characterized L-Lactate Oxidases

L-lactate oxidases have been characterized in numerous organisms:

  • Lentilactobacillus hilgardii (UniProt: C0XIJ3)
  • Aerococcus viridans (UniProt: Q44467)
  • Mycobacterium smegmatis (UniProt: O33655)
  • Lactobacillus plantarum
  • Pediococcus species

The enzyme is found across 161+ bacterial genera, indicating widespread distribution and functional importance.

The extensive characterization of L-lactate oxidases in a wide array of organisms, coupled with the experimental validation of its distinct enzymatic activity, provides strong evidence for the necessity of a specific GO term. The enzyme's presence in over 161 bacterial genera underscores its widespread distribution and functional significance in various metabolic pathways. Published literature, including detailed mechanistic studies and experimental validations, further supports its unique biochemical properties. By creating a specific GO term, we can accurately annotate these enzymes and reflect their importance in bacterial metabolism. This term will also aid in the annotation of proteins and metabolic pathways, ensuring that the scientific community can effectively track and analyze the function of L-lactate oxidase across diverse species.

Proposed Annotation Guidelines

When to Use This Term:

  • Enzymes that specifically oxidize L-lactate to pyruvate using O2 as an electron acceptor
  • FMN-dependent lactate oxidases producing H2O2
  • Proteins with demonstrated EC 1.1.3.2 activity

When NOT to Use This Term:

  • NAD+-dependent lactate dehydrogenases (use GO:0140171)
  • Lactate monooxygenases producing acetate (use GO:0050040)
  • D-lactate oxidases (would need a separate term)

These guidelines ensure the accurate application of the proposed GO term, preventing misannotation and improving the reliability of functional classifications.

Quality Control

This new term would improve annotation quality by:

  1. Reducing over-annotation with the general (S)-2-hydroxy-acid oxidase term
  2. Enabling precise functional annotation consistent with biochemical evidence
  3. Facilitating accurate metabolic pathway reconstruction
  4. Supporting comparative genomics studies of lactate metabolism

By introducing this term, we enhance the accuracy and reliability of annotations, leading to improved data interpretation and scientific insights.

Annotation Strategy

Automated Annotation Propagation

The proposed term includes an exact mapping (skos:exactMatch) to RHEA:55868, which will enable automatic propagation of annotations to existing proteins:

  • Current RHEA annotations: 2,065 UniProtKB entries are currently annotated with RHEA:55868
  • Immediate impact: Upon creation of this GO term, these 2,065 proteins would be eligible for GO annotation with "L-lactate oxidase activity"
  • Annotation evidence: These would receive IEA (Inferred from Electronic Annotation) evidence codes based on the RHEA-to-GO mapping

Expected Annotation Coverage

Based on the RHEA mapping alone, this new term would immediately provide precise functional annotation for over 2,000 bacterial proteins across diverse species including:

  • Lactic acid bacteria (Lactobacillus, Pediococcus, Leuconostoc)
  • Pathogenic bacteria (Streptococcus, Mycobacterium)
  • Industrial fermentation organisms
  • Human microbiome species

This represents a significant improvement in annotation specificity for a metabolically important enzyme class.

The annotation strategy leverages automated propagation through the exact mapping to RHEA:55868, ensuring broad and immediate coverage. The current annotation of 2,065 UniProtKB entries with RHEA:55868 means that these proteins would automatically be eligible for GO annotation with L-lactate oxidase activity, using IEA evidence codes. This strategy maximizes the impact of the new term by rapidly updating existing annotations and improving the functional characterization of numerous bacterial proteins. The expected annotation coverage spans diverse species, highlighting the term's broad applicability and its potential to enhance our understanding of bacterial metabolism across various ecological niches.

Term Submitter

This term request was created by an AI agent operated by @cmungall (ORCID 0000-0002-6601-2165). It was checked for correctness and completeness by @cmungall

Additional Notes

This term request is based on comprehensive curation of the haox gene (UniProt: C0XIJ3) in Lentilactobacillus hilgardii as part of the AI-assisted gene curation project. The mechanistic details and metabolic context have been thoroughly reviewed using both literature analysis and bioinformatic investigation.

For further details, see https://github.com/ai4curation/ai-gene-review/tree/main/genes/LENH9/haox

The proposed term fills a clear gap in GO's coverage of oxidoreductase activities and would benefit the annotation of bacterial metabolic enzymes across numerous species involved in fermentation, dairy production, and the human microbiome.

This new term request was generated as part of the ai-gene-review project for systematic improvement of GO annotations.