| Gene/protein target | Protein name | EC number | Reaction / biochemical role | Main substrates/products | Cofactors / partner components | Pathway context in *Methylorubrum extorquens* AM1 | Cellular localization | Key evidence notes | Key quantitative data | Key citations, URLs, dates |
|---|---|---|---|---|---|---|---|---|---|---|
| **gcvT** (UniProt **C5AUG2**; locus **MexAM1_META1p0622**) | Aminomethyltransferase; glycine cleavage system T protein (GcvT) | **2.1.2.10** | **Forward GCV direction:** transfers the aminomethyl group from aminomethylated H protein to THF, releasing **NH3** and generating **5,10-methylene-THF** while converting H-protein to the reduced form; part of the overall GCS conversion of **glycine + THF + NAD+ → 5,10-methylene-THF + CO2 + NH3 + NADH + H+**. **Reverse/rGCS overall:** participates in glycine synthesis from **5,10-methylene-THF + NH4+ + CO2 + reducing power → glycine + THF**. (pqac-00000008, pqac-00000009, pqac-00000011) | Forward: aminomethyl-H protein + THF → methylene-THF + NH3 + reduced H-protein. Overall forward GCV consumes glycine, THF, NAD+. Reverse overall rGCS consumes 5,10-CH2-THF, NH4+, CO2, reductant to make glycine + THF. (pqac-00000008, pqac-00000009, pqac-00000011) | Requires **tetrahydrofolate (THF)** as one-carbon acceptor and functions with GCS **P, H, and L** proteins; H protein carries the lipoyl-bound intermediate, L reoxidizes/reduces the H-protein cycle in the canonical system. In engineered systems, chemical reductants such as **DTT** can replace the L-protein reduction step. (pqac-00000008, pqac-00000011, pqac-00000013) | Links the **glycine cleavage system** to the **THF one-carbon pool**. In methylotrophs such as *M. extorquens* AM1, H4F/THF-linked C1 metabolism feeds assimilation pathways including the **serine cycle**; more broadly, GcvT is also a core enzyme for the **reductive glycine pathway** used in synthetic C1 assimilation. (pqac-00000005, pqac-00000008, pqac-00000011) | **Cytosolic** bacterial enzyme (inference from bacterial GCS/THF metabolism and lack of membrane association in cited pathway descriptions). (pqac-00000008, pqac-00000009, pqac-00000011) | **Identity verification:** literature support matches UniProt annotation for GcvT as the **aminomethyltransferase/T protein** of GCS, but organism-specific primary literature directly characterizing **AM1 gcvT/C5AUG2** is limited in the retrieved set. Review evidence confirms that *M. extorquens* AM1 uses H4F-dependent C1 metabolism tightly connected to the serine cycle, consistent with a cytosolic GcvT role in THF-linked one-carbon transfer. General GCS sources define the T-protein reaction and complex role; recent synthetic-biology studies use the same enzyme function in reverse GCS/rGlyP implementations. (pqac-00000005, pqac-00000008, pqac-00000009, pqac-00000011) | 2023 re-engineered rGCS data: overall glycine synthesis step **5,10-CH2-THF + NH4+ + CO2 + NADH → glycine + THF + NAD+** had **ΔrG' = -1.2 kJ/mol**; replacing the canonical reduction with **DTT** gave **ΔrG' = -8.0 kJ/mol** for the redesigned glycine synthesis step. Optimized system reached **~75 μM/min** glycine production at **60 μM** engineered H protein vs **<20 μM/min** with wild-type H; produced **15.5 mM (~1.2 g/L) glycine in 3.5 h**, with **78% carbon yield from formaldehyde** and **31% from CO2**; with gaseous CO2 under **0.2 MPa**, glycine reached **3.0 mM** (30% CO2) or **2.3 mM** (10% CO2). These numbers describe the GCS module that includes the T protein, not AM1-native enzyme kinetics specifically. (pqac-00000011, pqac-00000013, pqac-00000014) | Liu J, Zhang H, Xu Y, Meng H, Zeng A-P. *Nature Communications* (2023-05), https://doi.org/10.1038/s41467-023-38490-w (pqac-00000011, pqac-00000013, pqac-00000014); Wittmiß M et al. *The Plant Journal* (2020-05), https://doi.org/10.1111/tpj.14773 (pqac-00000008); Klein VJ et al. *Microorganisms* (2022-01), https://doi.org/10.3390/microorganisms10020220 (pqac-00000005); Patel M. thesis/manuscript on GCS/rGlyP (2016) describing T-protein function (pqac-00000009) |


*Table: This table summarizes the verified identity, enzymatic role, pathway context, localization, evidence base, and quantitative data relevant to functional annotation of *Methylorubrum extorquens* AM1 gcvT (UniProt C5AUG2). It is useful as a compact evidence-backed annotation aid, while noting the limited organism-specific primary literature directly on AM1 gcvT.*