| Evidence type | Finding (with quantitative values) | Experimental context | Source (paper + year + DOI URL) |
|---|---|---|---|
| Target identity / core annotation | **mtdA** in *Methylorubrum extorquens* AM1 encodes **MtdA**, a **strictly NADP-dependent methylene-H4MPT dehydrogenase** that also catalyzes oxidation of **methylene-H4F**; later literature uses the same enzyme in THF-pathway engineering and identifies the AM1 enzyme as **MtdA / Me-MtdA** consistent with UniProt **P55818** annotation (pqac-00000001, pqac-00000002, pqac-00000011) | Primary biochemical genetics in AM1; later heterologous pathway reconstruction in *E. coli* | Hagemeier et al., 2000, Eur J Biochem, https://doi.org/10.1046/j.1432-1327.2000.01413.x; Marx et al., 2003, J Bacteriol, https://doi.org/10.1128/jb.185.23.7160-7168.2003; Yishai et al., 2018, ACS Synth Biol, https://doi.org/10.1021/acssynbio.8b00131 |
| Reaction(s) catalyzed | Catalyzes dehydrogenation of **methylene-H4MPT → methenyl-H4MPT** with **NADP+** reduction; also catalyzes **methylene-H4F → methenyl-H4F** with NADP+. Reversible dehydrogenation of methylene-H4F is specifically noted; no cyclohydrolase activity is assigned to MtdA itself (cyclohydrolase is **FchA/Mch** in adjacent pathway steps) (pqac-00000023, pqac-00000025, pqac-00000026) | Enzyme purified from recombinant expression and interpreted in AM1 C1 metabolism pathway maps | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x; Marx et al., 2003, https://doi.org/10.1128/jb.185.23.7160-7168.2003 |
| Substrate specificity | Dual pterin specificity: best substrate is **methylene-H4MPT**; **methylene-H4F** is also accepted, but catalytic efficiency for methylene-H4F is reported to be **~20-fold lower** than for methylene-H4MPT (pqac-00000000, pqac-00000016, pqac-00000026) | Comparative biochemical characterization of recombinant/purified MtdA | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Cofactor specificity | **Strictly NADP dependent**; cell extracts of methanol-grown AM1 had **2.6 U/mg** NADP-dependent methylene-H4MPT dehydrogenase activity versus **0.6 U/mg** NAD-dependent activity, the latter attributable to MtdB rather than MtdA (pqac-00000002, pqac-00000013, pqac-00000028) | Native AM1 cell extracts grown on methanol; chromatographic separation of NADP- vs NAD-dependent activities | Marx et al., 2003, https://doi.org/10.1128/jb.185.23.7160-7168.2003; Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Specific activity / expression | Recombinant overexpression in *E. coli* yielded extract activities up to **~370 U/mg**; purified MtdA showed specific activity **~1100 U/mg** with **44% purification yield**; ~**20 mg** purified enzyme obtained from ~**2.5 g** wet cells (pqac-00000010, pqac-00000014) | Heterologous expression from cloned **mtdA** in *E. coli* and purification of recombinant protein | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Kinetic parameters | Reported apparent **Km** values for MtdA include **~20 µM** for methylene-H4MPT, **~30 µM** for methylene-H4F, **~30 µM** for NADP+ with methylene-H4MPT, and **~10 µM** for NADP+ with methylene-H4F; lower NADP+ Km values support MtdA as the main methylene-H4MPT-oxidizing enzyme (pqac-00000014, pqac-00000023, pqac-00000030) | Kinetic comparison table for purified MtdA and MtdB | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Oligomeric state / size | MtdA subunit mass reported as **32 kDa**; apparent native molecular mass **~90 kDa**, consistent with a multimeric soluble enzyme (pqac-00000000, pqac-00000014, pqac-00000033) | Purified protein characterization | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Biochemical optima / inhibition | **pH optimum ~6.0**, **temperature optimum ~45°C**, **isoelectric point ~7.2**; **no inhibition by NADPH** reported (pqac-00000014, pqac-00000024) | Purified recombinant MtdA biochemical characterization | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Localization evidence | Methylene-H4MPT dehydrogenase activity was recovered from the **ultracentrifugation supernatant**; the **membrane fraction lacked NAD(P)-dependent methylene-H4MPT dehydrogenase activity**, supporting **soluble/cytosolic localization** for MtdA (pqac-00000035, pqac-00000032, pqac-00000033) | Native AM1 extract fractionation and chromatographic purification from soluble fraction | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x |
| Pathway context | MtdA functions in the **H4MPT-linked formaldehyde oxidation pathway** and also interfaces with the **H4F/serine-cycle branch** by generating methenyl-/formyl-H4F for biosynthetic C1 metabolism; pathway diagrams label it as **NADP-dependent methylene-H4F/methylene-H4MPT dehydrogenase** (pqac-00000002, pqac-00000031, pqac-00000038) | Formaldehyde oxidation/detoxification model in methylotrophic growth | Marx et al., 2003, https://doi.org/10.1128/jb.185.23.7160-7168.2003 |
| Essentiality / genetics | **No null mutants** in **mtdA** could be obtained; reduced-activity mutants could not grow on methanol, indicating likely **essentiality** for central C1 metabolism and/or formyl-H4F supply (pqac-00000001, pqac-00000015, pqac-00000038) | Gene disruption / allelic exchange studies in AM1 | Hagemeier et al., 2000, https://doi.org/10.1046/j.1432-1327.2000.01413.x; Marx et al., 2003, https://doi.org/10.1128/jb.185.23.7160-7168.2003 |
| Overexpression phenotype | **mtdA overexpression ~7.4-fold** above wild type reduced methanol sensitivity but **did not restore growth on methanol** in the absence of MtdB, implying MtdA can support only **moderate formaldehyde flux** and may be limited in vivo by its reliance on **NADP** rather than NAD (pqac-00000012, pqac-00000027, pqac-00000036) | Physiological complementation/overexpression analysis in AM1 H4MPT-pathway mutants | Marx et al., 2003, https://doi.org/10.1128/jb.185.23.7160-7168.2003 |
| Modern applications / real-world implementation | AM1 **MtdA** has been repurposed in engineered THF-based C1-assimilation modules: in synthetic reductive glycine/formate-assimilation systems, **M. extorquens** enzymes **FTL/Fch/MtdA** enable reduction of formyl-H4F to methylene-H4F and support assembly of biomass precursors or methyl donors from formate/CO2 (pqac-00000011, pqac-00000017) | Heterologous pathway engineering in *E. coli* and other synthetic C1-assimilation platforms | Yishai et al., 2018, https://doi.org/10.1021/acssynbio.8b00131; Mohr et al., 2025, https://doi.org/10.1186/s12934-025-02674-4 |


*Table: This table compiles key functional-annotation evidence for Methylorubrum extorquens AM1 MtdA, including biochemical activity, specificity, kinetics, localization, and physiological relevance. It is useful as a compact evidence map linking the UniProt annotation to primary literature and recent applications.*