NAD-dependent malate dehydrogenase (EC 1.1.1.37) of the LDH/MDH superfamily. It is a soluble, cytoplasmic enzyme that catalyzes the reversible NAD(+)-linked oxidation of (S)-malate to oxaloacetate, generating NADH. The enzyme sits at the malate/oxaloacetate node of central carbon metabolism, providing the oxaloacetate-regenerating step of the tricarboxylic acid (TCA) cycle and contributing reducing equivalents to respiratory metabolism. In Pseudomonas putida KT2440, enzyme assays of cell-free extracts show strong specificity for NAD(+) over NADP(+) (~98% NAD+), consistent with a canonical TCA-cycle malate dehydrogenase. The protein adopts the two-domain LDH/MDH fold with an N-terminal NAD(P)-binding Rossmann domain and a C-terminal substrate-binding domain, with a conserved His proton-acceptor active site and Arg residues that recognize the substrate carboxylates.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Root-level molecular function term; correct but uninformative for an enzyme with a defined activity. Reason: The gene product is an enzyme so catalytic activity is true, but this is a very general parent of the specific malate dehydrogenase activity (GO:0030060) and carries no specific information. Retained as non-core. |
| GO:0004459 L-lactate dehydrogenase (NAD+) activity | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter over-propagation from the LDH/MDH superfamily; this protein is a malate dehydrogenase, not a lactate dehydrogenase. Reason: This IEA (TreeGrafter/PANTHER) annotation reflects an electronic mis-assignment within the shared LDH/MDH superfamily node. UniProt and InterPro (Malate_DH_type3, IPR011275) classify Q88Q44 as a malate dehydrogenase (EC 1.1.1.37, GO:0030060), and experimental assays in KT2440 measured malate dehydrogenase, not L-lactate dehydrogenase, activity. L-lactate dehydrogenase activity is not supported and is contradicted by the malate-specific function; this is an over-propagated electronic inference appropriate to remove. |
| GO:0006089 lactate metabolic process | IEA GO_REF:0000118 | REMOVE | Summary: TreeGrafter over-propagation paired with the erroneous L-lactate dehydrogenase activity call; the gene functions in the TCA cycle, not lactate metabolism. Reason: This process annotation derives from the same incorrect LDH-superfamily grafting as GO:0004459. The enzyme acts on malate/oxaloacetate in the TCA cycle, not on lactate. Over-propagated electronic inference, appropriate to remove. The correct process is tricarboxylic acid cycle (GO:0006099), captured by UniProt keyword but missing from GOA. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Correct high-level oxidoreductase parent; uninformative relative to the specific malate dehydrogenase activity. Reason: True but general parent of GO:0030060. Retained as non-core background. |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Correct intermediate-level parent describing NAD(P)-linked CH-OH oxidoreductases; more specific malate dehydrogenase activity is preferred as core. Reason: Accurately describes the enzyme class (NAD-linked CH-OH oxidoreductase) but is a parent of the specific GO:0030060 L-malate dehydrogenase (NAD+) activity. Retained as non-core. |
| GO:0030060 L-malate dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct core molecular function, matching EC 1.1.1.37 and the UniProt catalytic activity (RHEA:21432, (S)-malate + NAD+ = oxaloacetate + NADH + H+). Reason: This is the core, well-supported molecular function. The UniProt RHEA/EC mapping and InterPro Malate_DH_type3 domain agree, and KT2440 cell-free extract assays demonstrated strong NAD+-specific malate dehydrogenase activity. |
| GO:0030060 L-malate dehydrogenase (NAD+) activity | ISS GO_REF:0000024 | ACCEPT | Summary: ISS transfer of the core malate dehydrogenase activity from a characterized ortholog (UniProtKB:P61889); consistent with all other evidence. Reason: Sequence-similarity transfer of the core function from an experimentally characterized MDH ortholog. Concordant with the EC mapping, InterPro family, conserved active-site/binding residues, and KT2440 enzyme assay data. Represents the core function of the gene. |
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Download this section (compressed HTML)Q: Is mdh (PP_0654) the sole NAD-dependent malate dehydrogenase in P. putida KT2440, or do malate:quinone oxidoreductase (Mqo) isozymes and malic enzymes provide redundant routes through the malate node?
Experiment: Purify recombinant PP_0654 and determine steady-state kinetic constants (Km, kcat) for L-malate, oxaloacetate, NAD+ and NADH to confirm the canonical ordered Bi-Bi mechanism and quantify directional preference under physiological conditions.
Experiment: Construct a clean mdh (PP_0654) deletion and assess growth on TCA-cycle and gluconeogenic carbon sources, with 13C-metabolic flux analysis to test rerouting of malate via malic enzyme and the glyoxylate shunt.
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