| Evidence type | Key finding about pckA/PEPCK | Quantitative values | Experimental/analysis context | Source and URL | Notes/implications for in vivo direction/pathway |
|---|---|---|---|---|---|
| Biochemistry/mechanism | ATP-dependent PEPCK catalyzes reversible OAA + ATP ↔ PEP + ADP + CO2; requires Mg2+ and Mn2+ and proceeds via decarboxylation to an enolate intermediate before phosphoryl transfer. ATP-dependent forms are common in bacteria. (pqac-00000002, pqac-00000003, pqac-00000004) | ΔrG′m ≈ -6.8 ± 6.2 kJ/mol for ATP-dependent reaction; metal requirement includes Mg2+ with nucleotide and Mn2+ at active site. (pqac-00000003) | General enzyme biochemistry at the PEP-pyruvate-OAA node; not AM1-specific. | Rojas 2023 AoB Plants; Koendjbiharie 2021 FEMS Microbiol Rev. https://doi.org/10.1093/aobpla/plad053 ; https://doi.org/10.1093/femsre/fuaa061 | Supports annotation of UniProt C5B045 as ATP-dependent PEPCK acting at the central C3/C4 branchpoint; reaction can run either way depending on network demands. |
| Fluxomics | In Methylorubrum/Methylobacterium extorquens AM1 during methylotrophic growth, PEPCK is part of the dense C3/C4 interconversion subnetwork and functions as a C4→C3 step linked to PEP/OAA cycling and central CO2 release. (pqac-00000000, pqac-00000001) | PEPCK flux 0.26 mmol·g^-1·h^-1; malic enzyme 0.36 mmol·g^-1·h^-1; PEP/OAA cycling accounted for ~13% of recycled PEP; Me-THF assimilation flux 2.4 ± 0.02 mmol·g^-1·h^-1. (pqac-00000000) | AM1, methylotrophic growth; genome-scale reconstruction integrated with 13C-fluxomics. | Peyraud 2011 BMC Syst Biol. https://doi.org/10.1186/1752-0509-5-189 | Experimental evidence favors substantial in vivo OAA→PEP operation under methanol growth, contributing to substrate cycling/anaplerotic flexibility rather than being a dedicated sole gluconeogenic route. |
| Fluxomics | Earlier chemostat 13C-labeling work detected essentially no significant exchange through the combined PEPC/PEPCK step under the tested steady-state condition, indicating strong condition dependence of this node. (pqac-00000027) | Exchange coefficient for combined PEPC/PEPCK: 0 (0.19) in WT and 0 (0.16) in phaR mutant; dilution rate 0.09 h^-1 (~80% of µmax). (pqac-00000027) | AM1 methanol-limited chemostats; WT and phaR mutant. | Van Dien 2003 Biotechnol Bioeng. https://doi.org/10.1002/bit.10745 | Suggests pckA usage is context-sensitive; lack of exchange in one chemostat regime does not contradict active net OAA→PEP flux in other methylotrophic conditions. |
| Genetics/phenotype | 13C-MFA and knockout analysis show pck contributes more to biomass-yield-optimized metabolism than to maximal growth rate; PEPCK is active in LL under low cobalt but negligible in VL and in LL under high cobalt. (pqac-00000021, pqac-00000026) | LL + HY biomass yield 8.90 ± 0.59 g/mol; Δpck LL + HY 4.84 ± 0.98 g/mol (−4.07 g/mol; 54% of WT). VL + HY biomass yield 6.45 ± 0.49 g/mol; Δpck VL + HY 5.26 ± 0.52 g/mol (82% of WT). LL growth rate remained ~0.09–0.10 h^-1 with Δpck showing only small effect. (pqac-00000021, pqac-00000029) | AM1 LL and VL variants; HY vs HYC cobalt conditions; targeted Δpck mutant with 13C-MFA. | Fu 2016 BMC Microbiol. https://doi.org/10.1186/s12866-016-0778-4 | Strongest AM1-specific genetic evidence: pckA supports high biomass yield, especially in LL/low-cobalt conditions, consistent with OAA→PEP flux feeding efficient assimilatory C3/C4 balancing. |
| Genetics/phenotype | PEPCK consumes ATP, whereas alternative PK/PYRDH routes produce ATP/NADH; this energetic contrast helps explain why pck loss mainly lowers yield-associated metabolism rather than abolishing growth. (pqac-00000021, pqac-00000025, pqac-00000028) | Qualitative energetic comparison: PEPCK consumes 1 ATP. (pqac-00000025, pqac-00000028) | AM1 strain-comparison and mutant study under methanol growth with different cobalt levels. | Fu 2016 BMC Microbiol. https://doi.org/10.1186/s12866-016-0778-4 | Implies pckA participates in a higher-yield, more assimilatory C3/C4 strategy, while faster-growth states rely more on PK/PYRDH and less on PEPCK. |
| Modeling/engineering | Recent methanol-to-glycolate engineering/modeling in Methylorubrum extorquens identifies PCK as a candidate enzyme to strengthen the OAA→PEP link and potentially generate extra ATP instead of relying solely on PPC-centered solutions. (pqac-00000012, pqac-00000013) | 312,373 GA-forming EFMs computed; 267,347 analyzed; maximal theoretical GA yield 0.5 mol/mol methanol (1.19 g/g), growth-coupled near-maximal yield ~0.487 mol/mol; some EFMs co-produced up to 0.188–0.250 mol ATP/mol methanol; best fed-batch strain reached total 1.2 g/L glycolic + lactic acid. (pqac-00000013, pqac-00000015) | Engineered Methylorubrum extorquens TK 0001 for glycolic acid production; constraint-based modeling plus strain engineering. | Dietz 2024 Microb Cell Fact. https://doi.org/10.1186/s12934-024-02583-y | Although not direct AM1 pckA functional genetics, this recent work reinforces that the PEP/OAA node—and potentially PCK activity—is a practical engineering handle in methylotrophic central metabolism. |
| Comparative genomics/regulation | In type II methylotrophs, PEPC and other PEP-pyruvate-OAA node enzymes are broadly distributed, but the 2024 pangenome excerpt did not explicitly report pckA/PEPCK. For ATP-dependent bacterial PEPCKs more generally, specific allosteric regulation is known in some bacteria (e.g., Ca2+ activation in E. coli), but no AM1-specific regulator for pckA was identified in the gathered evidence. (pqac-00000006, pqac-00000017, pqac-00000019) | 75 type II methylotroph genomes analyzed; 256 exact core gene families identified; PEPC present across all 75 organisms, but no explicit pckA statistic reported in the extracted text. (pqac-00000017, pqac-00000019) | Comparative genomics across type II methylotrophs; broader ATP-PEPCK regulation review. | Samanta 2024 mSystems; Koendjbiharie 2021 FEMS Microbiol Rev. https://doi.org/10.1128/msystems.00248-24 ; https://doi.org/10.1093/femsre/fuaa061 | For AM1 annotation, regulation remains a gap: the protein is confidently assigned by sequence/family, but direct transcriptional/allosteric control in AM1 is not established by the retrieved evidence. |


*Table: This table summarizes experimentally supported and recent modeling evidence for Methylorubrum extorquens AM1 pck/pckA, emphasizing reaction chemistry, in vivo pathway role, mutant phenotypes, and biotechnology relevance. It is designed as a compact annotation aid linking each major claim to specific cited evidence.*