| Claim/feature | Evidence type | Key quantitative data | Source (first author year journal) and DOI/URL |
|---|---|---|---|
| **Target identity:** **glyA** in *Methylorubrum extorquens* AM1 (formerly *Methylobacterium extorquens*) encodes **serine hydroxymethyltransferase (SHMT; EC 2.1.2.1)**, a key serine-cycle enzyme | Genetics, comparative sequence | **glyA ORF = 1,305 bp**; predicted polypeptide **~46.3 kDa**; conserved SHMT motif **GGHLTHG**; reported as **single detectable copy** in AM1 (pqac-00000008, pqac-00000011) | **Chistoserdova 1994, Journal of Bacteriology**. DOI: 10.1128/jb.176.21.6759-6762.1994. URL: https://doi.org/10.1128/jb.176.21.6759-6762.1994 |
| **Primary enzymatic function:** SHMT catalyzes the reversible conversion between **serine + THF** and **glycine + 5,10-methylene-THF**; in AM1 physiological direction is serine formation from glycine + activated C1 unit | Biochemistry, pathway analysis | AM1 assays were performed in the **physiological direction** (serine formation from **glycine + C1 unit**); canonical SHMT reaction defined as THF-dependent serine/glycine interconversion (pqac-00000000, pqac-00000002, pqac-00000019) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001; **Drago 2023, Communications Chemistry**. DOI: 10.1038/s42004-023-00964-9. URL: https://doi.org/10.1038/s42004-023-00964-9 |
| **Cofactors and family assignment:** SHMT is a **PLP-dependent** enzyme that also requires a folate co-substrate for C1 transfer | Structure, enzymology | PLP forms an **internal aldimine with catalytic Lys** in solved SHMT structures; SHMT classified as a **PLP-dependent** enzyme across bacteria and eukaryotes (pqac-00000018, pqac-00000019, pqac-00000022) | **Drago 2023, Communications Chemistry**. DOI: 10.1038/s42004-023-00964-9. URL: https://doi.org/10.1038/s42004-023-00964-9; **Ma’ruf 2023, Amino Acids**. DOI: 10.1007/s00726-022-03205-w. URL: https://doi.org/10.1007/s00726-022-03205-w |
| **Native folate species in AM1:** the physiologically relevant C1 carrier is likely a **polyglutamylated folate**, not simple monoglutamyl THF | Biochemistry | **Tetrahydropteroyltriglutamate** stimulated serine synthesis more strongly than **tetrahydropteroylmonoglutamate**; AM1 identified natural C1 carrier as **tetrahydropteroyl-tetraglutamate** rather than simple THF in whole-pathway analysis (pqac-00000000, pqac-00000002, pqac-00000013) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001 |
| **Pathway role in methylotrophy:** GlyA is the **first enzyme of the serine cycle** and links H4F-linked C1 metabolism to formaldehyde assimilation and biosynthesis | Genetics, genomics, review | Supplies **methylene-H4F** for biosynthesis (e.g., purines) and participates directly in formaldehyde assimilation through the serine cycle (pqac-00000001, pqac-00000003, pqac-00000004, pqac-00000014) | **Chistoserdova 2003, Journal of Bacteriology**. DOI: 10.1128/JB.185.10.2980-2987.2003. URL: https://doi.org/10.1128/jb.185.10.2980-2987.2003; **Ochsner 2015, Applied Microbiology and Biotechnology**. DOI: 10.1007/s00253-014-6240-3. URL: https://doi.org/10.1007/s00253-014-6240-3 |
| **Mutant phenotype:** insertional **glyA null mutants lose SHMT activity** and **cannot grow on C1 compounds** | Genetics, enzymology | **No measurable SHMT activity** in glyA mutants; mutants **lost ability to grow on C1 compounds**, including **methanol**, even when supplemented with **glycine or serine** (pqac-00000010, pqac-00000011) | **Chistoserdova 1994, Journal of Bacteriology**. DOI: 10.1128/jb.176.21.6759-6762.1994. URL: https://doi.org/10.1128/jb.176.21.6759-6762.1994 |
| **Growth substrate specificity:** glyA is **not required for succinate growth**, but is required for methylotrophy and contributes to C2 metabolism | Genetics | glyA mutants **grew normally on succinate**; one report states mutant **lost ability to grow on C1 as well as C2 compounds** but still grew on succinate; glyoxylate chemically rescued some C2-growth defects (pqac-00000008, pqac-00000009, pqac-00000010) | **Chistoserdova 1994, Journal of Bacteriology**. DOI: 10.1128/jb.176.21.6759-6762.1994. URL: https://doi.org/10.1128/jb.176.21.6759-6762.1994 |
| **Chemical complementation insight:** **glyoxylate** can rescue growth on some **C2 substrates** but **not methanol**, implying a direct indispensable role for GlyA in C1 assimilation beyond glyoxylate supply | Genetics, physiology | **2–10 mM glyoxylate** supported growth on **ethanol or ethylamine**, but **up to 10 mM glyoxylate did not restore growth on methanol** (pqac-00000009, pqac-00000010) | **Chistoserdova 1994, Journal of Bacteriology**. DOI: 10.1128/jb.176.21.6759-6762.1994. URL: https://doi.org/10.1128/jb.176.21.6759-6762.1994 |
| **Potential flux bottleneck / rate-limiting step during methylotrophic growth** | Biochemistry, systems analysis | Measured **maximal GlyA activity ~30 mU mg⁻¹** (≈ **30 nmol min⁻¹ mg⁻¹**); calculated minimum needed for observed methanol-growth flux **~165 nmol min⁻¹ mg⁻¹**; estimated specific carbon-fixation demand **~330 nmol min⁻¹ mg⁻¹ protein**; measured activity therefore far below theoretical minimum (pqac-00000002, pqac-00000013, pqac-00000017) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001 |
| **Metabolite evidence for bottleneck:** elevated upstream intermediates are consistent with limited GlyA flux | Biochemistry, metabolomics interpretation | Reported intracellular **glyoxylate and glycine >0.10 mM**, consistent with buildup upstream of GlyA and with GlyA as a candidate control point (pqac-00000002, pqac-00000013) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001 |
| **Regulation by carbon source:** serine-cycle enzymes including GlyA are **induced on methanol** and down-regulated on nonrequired substrates | Biochemistry, proteomics, systems biology | Whole-pathway enzyme assays showed **strict differential regulation** depending on growth substrate; Figure-based summary indicates strong induction of serine-cycle enzymes on methanol (pqac-00000000, pqac-00000014, pqac-00000015) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001; **Laukel 2004, PROTEOMICS**. DOI: 10.1002/pmic.200300713. URL: https://doi.org/10.1002/pmic.200300713 |
| **Proteomic support for pathway assignment:** GlyA is detected as part of the methylotrophy-associated serine-cycle network | Proteomics | Proteome comparisons identified serine-cycle enzymes and explicitly note **serine hydroxymethyltransferase (GlyA)** among pathway components; GlyA is not genomically clustered with all serine-cycle genes (pqac-00000001) | **Laukel 2004, PROTEOMICS**. DOI: 10.1002/pmic.200300713. URL: https://doi.org/10.1002/pmic.200300713; **Chistoserdova 2003, Journal of Bacteriology**. DOI: 10.1128/JB.185.10.2980-2987.2003. URL: https://doi.org/10.1128/jb.185.10.2980-2987.2003 |
| **Role beyond methanol assimilation:** GlyA also intersects with glycine-generating pathways and broader one-carbon metabolism in related *Methylorubrum* physiology | Physiology, pathway genetics | In *M. extorquens* PA1 glycine betaine catabolism yields **glycine + methylene-THF**, which are stated to be used by **GlyA to make serine**, linking glycine handling to central metabolism (pqac-00000007) | **Hying 2024, Applied and Environmental Microbiology**. DOI: 10.1128/aem.02090-23. URL: https://doi.org/10.1128/aem.02090-23 |
| **Recent mechanistic update (2023): catalytic base assignment** | Structure, neutron/X-ray crystallography | Room-temperature joint neutron/X-ray structures support **Glu53** in bacterial TthSHMT (analogous **Glu98** in hSHMT2) as the **general base**, rather than His residues, for serine retro-aldol chemistry (pqac-00000018, pqac-00000019, pqac-00000020) | **Drago 2023, Communications Chemistry**. DOI: 10.1038/s42004-023-00964-9. URL: https://doi.org/10.1038/s42004-023-00964-9 |
| **Recent mechanistic update (2023): protonation states** | Structure, neutron crystallography | Direct H/D visualization showed **PLP pyridine N1 protonated**, **phenolic O3′ deprotonated**, **Schiff-base N non-protonated**; active-site histidines were **neutral/monoprotonated**, arguing against His as catalytic base (pqac-00000018, pqac-00000020) | **Drago 2023, Communications Chemistry**. DOI: 10.1038/s42004-023-00964-9. URL: https://doi.org/10.1038/s42004-023-00964-9 |
| **Recent mechanistic update (2024): folate binding and gating loop** | Structure | Folate analog binding induced **~4–5 Å gating-loop closure** and adjacent rearrangements; structures support a universal role for the conserved **active-site glutamate** in acid–base catalysis and show folate-pocket geometry relevant to substrate/cofactor access (pqac-00000021, pqac-00000023) | **Drago 2024, Chemical Science**. DOI: 10.1039/d4sc03187c. URL: https://doi.org/10.1039/d4sc03187c |
| **Localization / compartmentation inference:** no evidence for secretion or membrane localization; function is consistent with a **soluble cytosolic metabolic enzyme** | Inference from pathway biochemistry, assays | Activity measured in **cell extracts**; all evidence places GlyA in intracellular folate/serine-cycle metabolism; no periplasmic, membrane, or extracellular localization data were identified in the retrieved AM1 literature (pqac-00000000, pqac-00000010) | **Šmejkalová 2010, PLoS ONE**. DOI: 10.1371/journal.pone.0013001. URL: https://doi.org/10.1371/journal.pone.0013001; **Chistoserdova 1994, Journal of Bacteriology**. DOI: 10.1128/jb.176.21.6759-6762.1994. URL: https://doi.org/10.1128/jb.176.21.6759-6762.1994 |


*Table: This table summarizes the main functional-annotation claims for glyA/SHMT (UniProt P50435) in Methylorubrum extorquens AM1, integrating organism-specific genetics and biochemistry with recent 2023-2024 structural mechanism studies.*