mdh

Gene Description

mdh encodes NAD-dependent malate dehydrogenase (EC 1.1.1.37), which catalyzes the reversible oxidation of (S)-malate (L-malate) to oxaloacetate with concomitant reduction of NAD+ to NADH. This is a critical step in the tricarboxylic acid (TCA) cycle that regenerates oxaloacetate for continued TCA cycle operation. The enzyme belongs to the LDH/MDH superfamily, specifically the MDH type 3 family. In methylotrophs, Mdh plays a crucial role in completing the TCA cycle: carbon flowing from C1 compounds through the serine cycle → glycolysis → acetyl-CoA enters the TCA cycle via citrate synthase, and Mdh regenerates oxaloacetate from malate (produced by fumarase), closing the cycle. The oxaloacetate product can then react with another molecule of acetyl-CoA to continue the TCA cycle, or can be diverted to gluconeogenesis via pckA. The enzyme is NAD-dependent (not NADP-dependent like some other MDH isoforms) and functions in the cytoplasm. The reaction is reversible, allowing Mdh to function in both the oxidative direction (malate → oxaloacetate during TCA cycle flux) and the reductive direction (oxaloacetate → malate for anaplerotic or biosynthetic purposes). Mdh is essential for both energy production through complete oxidation of acetyl-CoA and for maintaining the supply of oxaloacetate needed for continued carbon assimilation.

Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0003824 catalytic activity	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: This is an extremely general parent term for all enzymes. While technically correct, the specific term GO:0030060 (L-malate dehydrogenase activity) provides precise functional annotation.
GO:0004459 L-lactate dehydrogenase (NAD+) activity	IEA GO_REF:0000118	REMOVE	Summary: This annotation is incorrect. This protein is malate dehydrogenase (EC 1.1.1.37), not lactate dehydrogenase (EC 1.1.1.27). The enzyme catalyzes the oxidation of malate to oxaloacetate, not lactate to pyruvate. This appears to be a misannotation by TreeGrafter. [file:METEA/mdh/mdh-uniprot.txt, "Malate dehydrogenase"; "(S)-malate + NAD(+) = oxaloacetate + NADH"]
GO:0006089 lactate metabolic process	IEA GO_REF:0000118	REMOVE	Summary: This annotation is incorrect. This enzyme metabolizes malate, not lactate. This appears to be a misannotation by TreeGrafter, likely due to the structural similarity between the LDH/MDH superfamily members. [file:METEA/mdh/mdh-uniprot.txt, "Malate dehydrogenase"; "(S)-malate + NAD(+) = oxaloacetate + NADH"]
GO:0006099 tricarboxylic acid cycle	IEA GO_REF:0000120	ACCEPT	Summary: Mdh catalyzes the reversible malate/oxaloacetate interconversion at the TCA cycle node. Falcon deep research notes that in canonical aerobic metabolism this enzyme functions in the citric acid cycle, while in M. extorquens AM1 grown on C1 compounds it operates at the serine-cycle/TCA interface, primarily reducing oxaloacetate. The 2024 systems-level study places mdh among coordinately regulated serine-cycle/H4F-pathway genes. Reason: Core biological process. The UniProt/HAMAP TCA cycle assignment is corroborated by organism-specific structural and systems-level evidence in the falcon deep research, which situates Mdh at the TCA/serine-cycle interface of central carbon metabolism. Supporting Evidence: file:METEA/mdh/mdh-deep-research-falcon.md in aerobic organisms MDH functions in the citric acid cycle file:METEA/mdh/mdh-deep-research-falcon.md Zhang et al. include mdh among serine-cycle/H4F-pathway-associated genes
GO:0016491 oxidoreductase activity	IEA GO_REF:0000120	KEEP AS NON CORE	Summary: This is a very general parent term for all oxidoreductases. While technically correct, the more specific term GO:0016616 provides better mechanistic annotation.
GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor	IEA GO_REF:0000002	ACCEPT	Summary: This accurately describes the enzymatic mechanism - Mdh is an oxidoreductase that acts on the CH-OH group of malate, using NAD+ as the electron acceptor. [file:METEA/mdh/mdh-uniprot.txt, "(S)-malate + NAD(+) = oxaloacetate + NADH"]. Falcon deep research confirms NAD(H) (not NADP) dependence from direct structural and assay evidence on the AM1 enzyme. Reason: Accurate mechanistic parent term. The NAD-dependence underlying this term is directly supported by the AM1-specific crystallographic and kinetic study summarized in the falcon deep research (NAD+ bound in the structure; NADH used as reductant). Supporting Evidence: file:METEA/mdh/mdh-deep-research-falcon.md The target protein is the NAD(H)-dependent malate dehydrogenase of Methylorubrum extorquens AM1
GO:0019752 carboxylic acid metabolic process	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: This is a very general parent term that is correct but not informative. Malate is a carboxylic acid, but more specific terms about TCA cycle would be more useful.
GO:0030060 L-malate dehydrogenase (NAD+) activity	IEA GO_REF:0000120	ACCEPT	Summary: This is the primary and specific catalytic activity of Mdh - the NAD-dependent reversible interconversion of (S)-malate and oxaloacetate. [file:METEA/mdh/mdh-uniprot.txt, "Malate dehydrogenase"; "(S)-malate + NAD(+) = oxaloacetate + NADH + H(+)"]. Falcon deep research provides direct AM1-specific support: a high-resolution crystal structure with bound ligands and kinetic measurements for oxaloacetate reduction, confirming NAD(H) dependence and the LDH/MDH superfamily Rossmann-fold architecture. Reason: Core molecular function. Directly confirmed for the AM1 protein (Q84FY8) by the Gonzalez 2018 structure/kinetics study summarized in the falcon deep research, which reports the oxaloacetate-reduction reaction, NAD(H) cofactor usage, and LDH/MDH-superfamily fold. Supporting Evidence: file:METEA/mdh/mdh-deep-research-falcon.md reversibly catalyzes the reduction of oxaloacetate to (2S)-malate using NADH as a reductant file:METEA/mdh/mdh-deep-research-falcon.md Km = 36.8 ± 0.6 mM for oxaloacetate and kcat = (4.6 ± 0.1) × 10^2 s^-1

Core Functions

Mdh catalyzes the NAD-dependent reversible oxidation of (S)-malate (L-malate) to oxaloacetate with concomitant reduction of NAD+ to NADH. This is a critical step in the TCA cycle that regenerates oxaloacetate for continued cycle operation. In the context of methylotrophic metabolism, Mdh completes the TCA cycle: carbon flowing from C1 compounds through the serine cycle → glycolysis → acetyl-CoA enters the TCA cycle via citrate synthase, and Mdh regenerates oxaloacetate from malate (produced by fumarase), closing the cycle. The oxaloacetate product can then react with another molecule of acetyl-CoA (via citrate synthase) to continue the TCA cycle, or can be diverted to gluconeogenesis via pckA. The enzyme belongs to the LDH/MDH superfamily (MDH type 3 family) and functions in the cytoplasm. The reaction is reversible, allowing Mdh to function in both oxidative (malate → oxaloacetate) and reductive (oxaloacetate → malate) directions depending on metabolic needs. Mdh is essential for both energy production and maintaining the supply of oxaloacetate for carbon assimilation.

Molecular Function:

L-malate dehydrogenase (NAD+) activity

Directly Involved In:

tricarboxylic acid cycle

Supporting Evidence:

file:METEA/mdh/mdh-uniprot.txt
Catalyzes the reversible oxidation of malate to oxaloacetate.
file:METEA/mdh/mdh-uniprot.txt
(S)-malate + NAD(+) = oxaloacetate + NADH + H(+)
file:METEA/mdh/mdh-deep-research-falcon.md
reversibly catalyzes the reduction of oxaloacetate to (2S)-malate using NADH as a reductant
file:METEA/mdh/mdh-deep-research-falcon.md
in *M. extorquens* AM1 during growth on C1 compounds it functions in the serine pathway by primarily reducing oxaloacetate

Gene Description

Existing Annotations Review

Core Functions

References

Tags

Deep Research

Falcon

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