| Claim/Field | Evidence summary | Organism/strain context | Year | Source (DOI/URL) | Citation id(s) |
|---|---|---|---|---|---|
| Gene/protein identifiers | Target identity is consistent across sources as **Mdh**, locus **PP_0654**, in *Pseudomonas putida* KT2440/EM42-derived strains; recent proteomics papers explicitly annotate **Mdh (PP_0654)**, matching UniProt **Q88Q44** and the annotation “probable malate dehydrogenase.” | *P. putida* KT2440 and derivatives (including EM42-derived xylose-engineered strains) | 2015, 2023, 2024 | Nikel et al., JBC, 2015, https://doi.org/10.1074/jbc.m115.687749; Dvořák et al., bioRxiv, 2023, https://doi.org/10.1101/2023.05.19.541448; Dvořák et al., Nat Commun, 2024, https://doi.org/10.1038/s41467-024-46812-9 | (pqac-00000000, pqac-00000003, pqac-00000006) |
| Reaction/EC | By family annotation and authoritative MDH review, the enzyme catalyzes the reversible reaction **L-malate + NAD+ ⇌ oxaloacetate + NADH + H+** (**EC 1.1.1.37**). The 2024 review describes canonical MDH chemistry as NAD+-dependent, ordered Bi-Bi, with strong specificity for L-malate/oxaloacetate. | General MDH definition applied to bacterial Mdh; consistent with *P. putida* PP_0654 annotation | 2024 | de Lorenzo et al., Essays Biochem, 2024, https://doi.org/10.1042/EBC20230086 | (pqac-00000008, pqac-00000009, pqac-00000010, pqac-00000011) |
| Cofactor specificity (quantitative) | In *P. putida* KT2440 cell-free extracts, **Mdh (PP_0654)** is strongly **NAD+-specific**. Under saturating conditions: **98.4 ± 0.7% NAD+** vs **1.6 ± 0.7% NADP+**. Under quasi in vivo/non-saturating conditions: **97.5 ± 8.4% NAD+** vs **2.5 ± 1.2% NADP+**. | *P. putida* KT2440, exponential phase, M9 + 20 mM glucose, enzyme assays from cell-free extracts | 2015 | Nikel et al., JBC, 2015, Table 3, https://doi.org/10.1074/jbc.m115.687749 | (pqac-00000000, pqac-00000001, pqac-00000007) |
| Pathway roles | Evidence places Mdh at the **malate/oxaloacetate node of central carbon metabolism**, principally the **TCA cycle**, with links to **glyoxylate shunt engagement** and **redox balancing**. 13C-MFA during xylose growth identified high dehydrogenase flux including MDH and an active glyoxylate shunt; proteomics in evolved xylose strains showed Mdh abundance changes consistent with altered TCA use. In indigoidine-engineering strains, deletion/loss of **Mdh/PP_0654** rerouted malate away from oxaloacetate toward pyruvate while glyoxylate shunt proteins increased. | *P. putida* EM42-derived xylose strains (PD310, PD584, PD584 L3, PD689 tt L1); KT2440-derived indigoidine strains | 2023, 2024 | Dvořák et al., bioRxiv, 2023, https://doi.org/10.1101/2023.05.19.541448; Dvořák et al., Nat Commun, 2024, https://doi.org/10.1038/s41467-024-46812-9; Eng et al., bioRxiv, 2023, https://doi.org/10.1101/2023.03.16.532821 | (pqac-00000002, pqac-00000003, pqac-00000004, pqac-00000005, pqac-00000006) |
| Systems/engineering contexts | **Xylose adaptation:** MDH was tracked by **13C-metabolic flux analysis** and **proteomics** as part of rewiring central metabolism for growth on non-native D-xylose; Mdh abundance decreased slightly in one evolved lineage and increased in another, indicating alternative adaptation routes. **Indigoidine production:** **proteomics** showed **Mdh/PP_0654 had zero protein counts** in engineered deletion backgrounds, supporting deliberate rerouting of the malate→oxaloacetate step to improve product coupling. | Xylose-adapted EM42 derivatives; para-coumarate/indigoidine KT2440 engineering backgrounds | 2023, 2024 | Dvořák et al., bioRxiv, 2023, https://doi.org/10.1101/2023.05.19.541448; Dvořák et al., Nat Commun, 2024, https://doi.org/10.1038/s41467-024-46812-9; Eng et al., bioRxiv, 2023, https://doi.org/10.1101/2023.03.16.532821 | (pqac-00000002, pqac-00000003, pqac-00000004, pqac-00000005, pqac-00000006) |
| Localization inference | No direct localization experiment for PP_0654 was retrieved here. For bacterial central carbon MDH, the evidence supports a **cytosolic/cytoplasmic enzyme** acting in soluble metabolism rather than a membrane or periplasmic oxidoreductase. This is an **inference** from canonical MDH function, assay context using cell-free extracts, and pathway placement; localization should therefore be treated as probable rather than directly demonstrated in the cited *P. putida* papers. | *P. putida* KT2440/derivatives; inference from bacterial central metabolism | 2015, 2024 | Nikel et al., JBC, 2015, https://doi.org/10.1074/jbc.m115.687749; de Lorenzo et al., Essays Biochem, 2024, https://doi.org/10.1042/EBC20230086 | (pqac-00000000, pqac-00000010, pqac-00000011) |


*Table: This table summarizes the strongest evidence supporting the functional annotation of Pseudomonas putida KT2440 Mdh (PP_0654; UniProt Q88Q44). It integrates foundational biochemical evidence with 2023-2024 systems studies showing how the enzyme participates in central metabolism and metabolic engineering contexts.*