Epimerase (Msed_0639) and mutase (Msed_0638 and Msed_2055) convert (S)-methylmalonyl-coenzyme A (CoA) to succinyl-CoA in the Metallosphaera sedula 3-hydroxypropionate/4-hydroxybutyrate cycle

Abstract

Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [T(opt)], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B(12)-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO(2) fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α(2)β(2)) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni(2+), Co(2+), and Mg(2+)), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B(12)-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles.

Fig 1

Proposed autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula. (A) Enzymes ACC, MCR, MSR, HPCS, HPCD, ACR, MCE, MCM, SSR, HBCS, HBCD, CCH, and ACK (see Table 1). (B) Heat plot showing ACC (Msed_0147, Msed_0148, Msed_1375), MCE (Msed_0639), and MCM (Msed_0638, Msed_2055) under autotrophic (columns A) and heterotrophic (columns H) conditions. Normalized transcription levels are depicted in grayscale, with darker shading correlating with higher transcription levels.

Fig 2

Molecular assembly of recombinant M. sedula MCE and quaternary structure analysis. (A) The recombinant MCE purified by heat treatment and IMAC was analyzed by SDS-PAGE. (B) The quaternary structure of MCE was assayed by using size exclusion chromatography (Superdex 75).

Fig 3

Molecular assembly of M. sedula MCM and quaternary structure analysis. (A) Size exclusion chromatography (Superdex 200) of recombinant MCM purified through heat treatment and IMAC. (B) Basic native PAGE of recombinant coexpressed MCM. (C) Acidic native PAGE of recombinant coexpressed MCM (no M_r ladder available). (D) SDS-PAGE of recombinant coexpressed MCM purified through heat treatment and IMAC. (E and F) SDS-PAGE of elution peaks for recombinant coexpressed MCM from size exclusion chromatography.

Fig 4

Phylogenetic tree of characterized MCE. The organisms include Propionibacterium shermanii (P. shermanii), Pyrococcus horikoshii (P. horikoshii), Metallosphaera sedula DSM 5348 (M. sedula), Thermoanaerobacter tengcongensis (T. tengcongensis), Caenorhabditis elegans (C. elegans), Homo sapiens (H. sapiens), and Rattus norvegicus (R. norvegicus).

Fig 5

Phylogenetic tree of characterized MCM. The organisms include Metallosphaera sedula (M. sedula), Sinorhizobium meliloti (S. meliloti), Propionibacterium shermanii (P. shermanii), Methylobacterium extorquens (M. extorquens), Streptomyces cinnamonensis (S. cinnamonensis), Escherichia coli (E. coli), Homo sapiens (H. sapiens), Mus musculus (M. musculus), and Euglena gracilis Z (E. gracilis).

Fig 6

Conversion of (R,S)-methylmalonyl–CoA to succinyl-CoA by M. sedula MCE and MCM at 75°C and pH 7.0. From bottom to top: succinyl-CoA; methylmalonyl-CoA; formation of succinyl-CoA from (R,S)-methylmalonyl-CoA catalyzed by MCM; and formation of succinyl-CoA from (R,S)-methylmalonyl-CoA, with the S-isomer present first epimerized to the R-isomer by MCE.