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

Appl Environ Microbiol. 2012 Sep;78(17):6194-202. doi: 10.1128/AEM.01312-12. Epub 2012 Jun 29.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acyl Coenzyme A / metabolism*
  • Autotrophic Processes
  • Biotransformation
  • Carbon Dioxide / metabolism
  • Cobamides / metabolism
  • Escherichia coli / genetics
  • Gene Expression
  • Gene Expression Profiling
  • Heterotrophic Processes
  • Hydroxybutyrates / metabolism*
  • Methylmalonyl-CoA Mutase / chemistry
  • Methylmalonyl-CoA Mutase / genetics
  • Methylmalonyl-CoA Mutase / metabolism*
  • Protein Multimerization
  • Racemases and Epimerases / chemistry
  • Racemases and Epimerases / genetics
  • Racemases and Epimerases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sulfolobaceae / enzymology*
  • Sulfolobaceae / genetics
  • Sulfolobaceae / metabolism*

Substances

  • Acyl Coenzyme A
  • Cobamides
  • Hydroxybutyrates
  • Recombinant Proteins
  • methylmalonyl-coenzyme A
  • Carbon Dioxide
  • succinyl-coenzyme A
  • Racemases and Epimerases
  • methylmalonyl-coenzyme A racemase
  • Methylmalonyl-CoA Mutase