Fig 1.
The central metabolism of M. alcaliphilum 20Z.
1 –hexulose 6-phosphate synthase, 2 –phosphohexuloisomerase 3 –PPi-dependent 6-phosphofructokinase, 4 –glyceraldehyde-3-phosphate dehydrogenase, 5 –pyruvate dehydrogenase complex, 6 –citrate synthase, 7 –aconitase, 8 –isocitrate dehydrogenase, 9 – α-ketoglutarate dehydrogenase complex, 10 –succinyl CoA synthetase, 11 –succinate dehydrogenase, 12 –fumarase, 13 –malate dehydrogenase, 14 –malic enzyme, 15 –pyruvate carboxylase, 16 –PPi-dependent phosphoenolpyruvate carboxykinase, 17 –adenylosuccinate synthase, 18 –adenylosuccinate lyase, 19 –argininosuccinate synthase, 20 –argininosuccinate lyase, 21 – α-ketoglutarate: ferredoxin oxidoreductase, 23 –pentose phosphate pathway.
Fig 2.
Purification and determination of oligomeric state of the recombinant FumI and FumC from M. alcaliphilum 20Z.
(A) The purity of the proteins was determined by 12% SDS-PAGE. M, molecular mass of the markers. (B) Molecular mass determination by gel filtration chromatography.
Fig 3.
Effect of pH (A, B) and temperature (C) on the activity of FumI (solid line) and Fum C (dashed line) from M. alcaliphilum 20Z in the reactions of fumarate hydration (A, C) and malate dehydration (B): (diamond)–Tris-HCl, (square)–K-phosphate, (triangle)–Glycine-NaOH, (x)–MES-NaOH, (circle)–CHES-NaOH.
Table 1.
Kinetic parameters of M. alcaliphilum fumarases.
Fig 4.
The model structure of FumI homodimer from M. alcaliphilum obtained by AlphFold [18].
The N-terminal (alpha-type catalytic) domain is light blue, C-terminal (beta-type catalytic) domain is light green. The iron–sulfur cluster was determined according to the structure of fumarase 2 from Leishmania major (PDB ID 5L2R). Cys residues are red.
Fig 5.
An increase in the concentration of extracellular metabolites in the triple mutant strain 20Z-3E compared to the wild type during the growth on methanol (A) and methane (B). PC–pyruvate carboxylase, PPi-PEPCK–PPi-dependent phosphoenolpyruvate carboxykinase.
Table 2.
Analysis of the culture fluid of the mutant and WT strains of M. alcaliphilum growing on methane (μmol g-1 DCW).
Fig 6.
Phylogenetic tree of class I fumarases.
Tree topography and evolutionary distances are given by the neighbor-joining method with Poisson correction. The characterized enzymes are in bold [1, 2, 5, 15, 26, 27]. The amino acid accession numbers in the GeneBank are in brackets. Alphaproteobacterial methanotrophs are brown, gammaproteobacterial methanotrophs are violet, bacteria with FumA are orange, bacteria with FumB are blue, and bacteria with FumD are green.