title: 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.
authors: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.
authors: Yejun
authors: Y
authors: Han
authors: Aaron S
authors: AS
authors: Hawkins
authors: Michael W W
authors: MW
authors: Adams
authors: Robert M
authors: RM
authors: Kelly
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D052818:Autotrophic Processes; D001711:Biotransformation; D002245:Carbon Dioxide / Q000378:metabolism; D003038:Cobamides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D052836:Heterotrophic Processes; D006885:Hydroxybutyrates / Q000378:metabolism*; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016943:Sulfolobaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D003038:Cobamides; D006885:Hydroxybutyrates; D011994:Recombinant Proteins; C015357:methylmalonyl-coenzyme A; D002245:Carbon Dioxide; C012046:succinyl-coenzyme A; D019998:Racemases and Epimerases; C021581:methylmalonyl-coenzyme A racemase; D008765:Methylmalonyl-CoA Mutase