| Component | Enzyme name (EC) | Reaction step / role | Key cofactors / prosthetic groups | Evidence type | Key quantitative data (if any) | Notes for *Methylorubrum extorquens* AM1 context | Primary sources (DOI URL, year) |
|---|---|---|---|---|---|---|---|
| GcvP | Glycine dehydrogenase (decarboxylating) / glycine decarboxylase P-protein (EC 1.4.4.2) | First step of glycine cleavage: decarboxylates glycine, releasing CO2 and generating aminomethylated/lipoylated H-protein intermediate (Hint) from oxidized H-protein (Hox) | PLP is required/associated with P-protein; acts with lipoylated H-protein as acceptor of the aminomethyl moiety | Mechanistic in vitro; pathway engineering | In reconstituted GCS, omission of P-protein reduced glycine-cleavage activity to 10.34% of reference and glycine-synthesis activity to 34.07%; omission of both P-protein and PLP abolished both directions; reference cleavage rate 22.48 ± 3.47 μM HCHO·min⁻¹ and synthesis rate 5.95 ± 0.13 μM glycine·min⁻¹ (pqac-00000012, pqac-00000013) | UniProt C5AUG0 in AM1 is annotated as this conserved family member. Direct AM1-specific biochemical characterization was not retrieved, so function is inferred from conserved bacterial GCS chemistry consistent with the annotation (pqac-00000011, pqac-00000012) | Xu et al., https://doi.org/10.1101/2021.03.28.437365, 2021 (pqac-00000011, pqac-00000012, pqac-00000013, pqac-00000014) |
| GcvT | Aminomethyltransferase / T-protein (EC 2.1.2.10) | Second step: releases NH3 and transfers the one-carbon unit from Hint to THF to form 5,10-methylene-THF, leaving reduced H-protein (Hred) | THF is the one-carbon acceptor; reaction depends on aminomethyl transfer to folate pool | Mechanistic in vitro; pathway engineering | Omission of T-protein reduced glycine-cleavage activity to 51.91% and glycine-synthesis activity to 76.53%; omission of THF reduced activities more severely to 3.88% and 8.94%, respectively (pqac-00000012, pqac-00000014) | In methylotrophic alphaproteobacteria such as *Methylorubrum*, this step is relevant because it feeds 5,10-CH2-THF into C1 metabolism; direct AM1 gcvT/gcvP operon-level evidence was not retrieved here (pqac-00000011) | Xu et al., https://doi.org/10.1101/2021.03.28.437365, 2021 (pqac-00000011, pqac-00000012, pqac-00000014) |
| GcvH | Glycine cleavage system H-protein / aminomethyl carrier H-protein (no EC assigned as carrier protein) | Lipoyl-armed carrier that shuttles reaction intermediates among P-, T-, and L-proteins; cycles through oxidized (Hox), aminomethylated (Hint), and reduced (Hred) states | Covalently attached lipoic acid on conserved Lys64; lipoyl arm is central prosthetic group | Mechanistic in vitro; pathway engineering | H-protein is essential in vitro; no reaction without Hox. Lipoylated H-protein alone could support glycine synthesis and, with FAD, glycine cleavage in vitro; full GCS kcat ~0.01 s⁻¹ versus H-protein-alone ~0.0057 s⁻¹ in the reported system (pqac-00000006, pqac-00000007) | Although not specific to AM1, the conserved requirement for lipoylated GcvH supports annotation of C5AUG0 as part of a canonical bacterial glycine cleavage system rather than an unrelated protein (pqac-00000012, pqac-00000013) | Xu et al., https://doi.org/10.1101/2021.03.28.437365, 2021 (pqac-00000006, pqac-00000007, pqac-00000012, pqac-00000013) |
| GcvL | Dihydrolipoyl dehydrogenase / L-protein (EC 1.8.1.4) | Third step: reoxidizes reduced H-protein (Hred) to Hox, coupling electron transfer to NAD+ reduction | FAD-associated flavin chemistry and NAD+/NADH redox pair | Mechanistic in vitro; pathway engineering | Omission of L-protein reduced glycine-cleavage activity to 37.55% and glycine-synthesis activity to 74.78%; in H-protein-only cleavage assays, 40 μM FAD enabled activity with 5 mM NAD+ (pqac-00000012, pqac-00000013, pqac-00000014) | For AM1, GcvL is expected to be the redox-recycling partner of GcvP/H/T in the cytosolic bacterial GCS; no direct AM1-specific localization experiment was found in retrieved evidence (pqac-00000011, pqac-00000013) | Xu et al., https://doi.org/10.1101/2021.03.28.437365, 2021 (pqac-00000011, pqac-00000012, pqac-00000013, pqac-00000014) |
| GcvP/GcvT/GcvH/GcvL system | Reversible glycine cleavage system (overall pathway module) | Overall reversible reaction: glycine ⇌ CO2 + NH3 + 5,10-CH2-THF-linked C1 transfer; in the reverse direction, forms glycine from NH4+, CO2, and 5,10-CH2-THF equivalents | PLP, lipoyl-H, THF, FAD/NAD(H) | Pathway engineering | In a 2023 chemoenzymatic rGCS platform using methanol + air-captured CO2, glycine reached 17.1 mM (1.3 g/L) in 5.5 h with 86% yield based on formaldehyde and 34% on CO2; from methanol + air-captured CO2, glycine reached 13.2 mM (1.0 g/L) in 5 h, serine 7.5 mM (0.8 g/L) in 8 h, and pyruvate 6.9 mM (0.6 g/L) in 5 h (pqac-00000016, pqac-00000017) | These engineering data are not from AM1, but they illustrate why GcvP-family enzymes are of current interest in C1 biomanufacturing and support the importance of the conserved reaction assigned to C5AUG0 (pqac-00000016, pqac-00000017) | Liu et al., https://doi.org/10.1038/s41467-023-38490-w, 2023 (pqac-00000016, pqac-00000017) |
| Glycine/C1 metabolism context | Intersection with glycine betaine catabolism and methylotrophy | In *Methylorubrum extorquens* physiology, glycine betaine degradation yields formaldehyde and glycine; formaldehyde enters methylotrophic metabolism and glycine can enter central metabolism via serine/pyruvate routes | THF-linked C1 metabolism and serine/glycine network implied; no direct cofactor assignment to AM1 GcvP in this physiology paper | Organism physiology | *M. extorquens* PA1 could not use glycine betaine as sole carbon source in wild type, whereas AM1 was reported as able to use glycine betaine as sole carbon and energy source; in PA1 experiments, growth assays used 8 mM glycine betaine (pqac-00000008) | This is the closest organism-specific evidence linking *Methylorubrum* glycine metabolism to C1 metabolism, but it does not directly characterize AM1 gcvP/C5AUG0; thus AM1 pathway assignment for gcvP remains inference-backed rather than directly tested here (pqac-00000008) | Hying et al., https://doi.org/10.1128/aem.02090-23, 2024 (pqac-00000008) |


*Table: This table summarizes the conserved functional annotation of bacterial GcvP and its partner proteins in the glycine cleavage system, with emphasis on evidence relevant to UniProt C5AUG0 from *Methylorubrum extorquens* AM1. It distinguishes direct mechanistic evidence from broader pathway-engineering and organism-physiology context, which is important because AM1-specific experimental literature for C5AUG0 was limited.*