Fig 1.
Reactions catalyzed by class I fumarases: (A) fumarase reaction, (B) (2S,3S)-tartrate dehydratase reaction, and (C) mesaconase reaction.
Fig 2.
Metabolic pathways involving mesaconase reaction: (A) mesaconate utilization, (B) methylaspartate pathway of glutamate fermentation.
Enzymes: 1, mesaconase; 2, succinyl-CoA:(S)-citramalate CoA-transferase; 3, (S)-citramalyl-CoA lyase; 4, glutamate mutase; 5, methylaspartate ammonia-lyase; 6, citramalate lyase.
Fig 3.
SDS-PAGE (12.5%) of fractions obtained during purification of recombinant fumarases FumA (A), FumB (B) and FumC (C) from E. coli K-12 as well as of a recombinant mesaconase/fumarase FumD from E. coli O157:H7 (ATCC 700728) (D).
M, molecular mass standard proteins; lane 1, cell extract of E. coli producing the corresponding proteins; lane 2, membrane fraction; lane 3, flow through after Ni-NTA column; lane 4, elution with 25 mM imidazole; lane 5, elution with 50 mM imidazole; lane 6, elution with 300 mM imidazole; lane 7 with 500 mM imidazole. The fractions eluted with 300 mM imidazole were used for the following enzyme characterization. Proteins were stained with Coomassie blue. The predicted molecular mass of native FumA is 60.3 kDa, that of FumB 60.1 kDa, that of FumC 50.5 kDa, and that of FumD 60.1 kDa.
Table 1.
Catalytic properties of recombinant fumarases from E. coli K-12 W3110.
Fig 4.
Co-localization of the genes for methylaspartate ammonia-lyase and glutamate mutase with the gene(s) for class I fumarase (mesaconase).
GntR, a putative transcriptional regulator for glutamate degradation; GlmS and GlmE, subunits of glutamate mutase; MutL (sometimes designated also as GlmL), a chaperone involved in reactivation of glutamate mutase [46]; Mal, methylaspartate ammonia-lyase; MesAB, α- and β-subunits of a putative mesaconase; CitDEF and CitCXG, subunits of citramalate lyase and enzymes involved in its biosynthesis/activation (named after the subunits of homologous citrate lyase [47,48]); MurI, glutamate racemase; HgdBC, subunits of 2-hydroxyglutaryl-CoA dehydratase; SfcA, malate dehydrogenase, decarboxylating.
Fig 5.
Co-localization of the genes for methylaspartate ammonia-lyase and glutamate mutase with mesaconase gene in Enterobacteriaceae.
GltP, glutamate transporter; FumD, promiscuous mesaconase/fumarase; CobA, cob(I)alamin adenosyltransferase; LTTR, a putative LysR family transcriptional regulator; GlsA, glutaminase; SumT, uroporphyrinogen-III C-methyltransferase; for the other abbreviations, see Fig 4. Note that the gene encoding a protein of unknown function (DUF1446) downstream of mal is often annotated as methylaspartate ammonia-lyase, for unknown reason. A homologous gene is present downstream of mal in Dictyoglomus thermophilum (Fig 4). Note also that glutaminase is known to contribute to the acid resistance that is an important feature for enterobacterial virulence [49,50].
Table 2.
Catalytic properties of recombinant fumarase D (ECs0757; ECATCC700728_0849) from E. coli O157:H7 (ATCC 700728).
Fig 6.
Phylogenetic tree of class I fumarases and related proteins.
The tree is based on amino acid sequence analysis of N-terminal part of class I fumarases consisting of one subunit (marked with”*” on the tree) and α-subunits of heterodimeric fumarases/ mesaconases/ (2R,3R)-tartrate dehydratases. (2R,3R)-Tartrate dehydratases are shown in blue, enzymes functioning in vivo as mesaconases in red (functions confirmed experimentally or based on the co-localization with glutamate fermentation genes). Tree topography and evolutionary distances are given by the neighbor-joining method with Poisson correction. The scale bar represents a difference of 0.1 substitutions per site. Numbers at nodes indicate the percentage bootstrap values for the clade of this group in 1000 replications. Only values above 70% were considered.