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 B12-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 CO2 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 (α2β2) 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 (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-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.

[1]  R. Stjernholm,et al.  The isolation, purification, and properties of methylmalonyl racemase. , 1963, The Journal of biological chemistry.

[2]  K. Stetter,et al.  Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation , 1999, Journal of bacteriology.

[3]  T. Bobik,et al.  Identification of the Human Methylmalonyl-CoA Racemase Gene Based on the Analysis of Prokaryotic Gene Arrangements , 2001, The Journal of Biological Chemistry.

[4]  D. Liang,et al.  Crystal structure of a putative methylmalonyl‐coenzyme a epimerase from Thermoanaerobacter tengcongensis at 2.0 Å resolution , 2009, Proteins.

[5]  K. Stetter,et al.  Metallosphaera sedula gen, and sp. nov. Represents a New Genus of Aerobic, Metal-Mobilizing, Thermoacidophilic Archaebacteria , 1989 .

[6]  R. Banerjee,et al.  IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone* , 2009, The Journal of Biological Chemistry.

[7]  R. Allen,et al.  Isolation and characterization of DL-methylmalonyl-coenzyme A racemase from rat liver. , 1985, Archives of biochemistry and biophysics.

[8]  R. Matthews,et al.  How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase. , 1994, Science.

[9]  G. Fuchs,et al.  Malonic Semialdehyde Reductase, Succinic Semialdehyde Reductase, and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales , 2009, Journal of bacteriology.

[10]  W. Yue,et al.  Structures of the Human GTPase MMAA and Vitamin B12-dependent Methylmalonyl-CoA Mutase and Insight into Their Complex Formation* , 2010, The Journal of Biological Chemistry.

[11]  Marc A. Martí-Renom,et al.  Tools for comparative protein structure modeling and analysis , 2003, Nucleic Acids Res..

[12]  M. Weiß,et al.  Identification of Missing Genes and Enzymes for Autotrophic Carbon Fixation in Crenarchaeota , 2010, Journal of bacteriology.

[13]  G. Fuchs,et al.  Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation. , 2003, European journal of biochemistry.

[14]  P. Kamoun,et al.  Valine is a precursor of propionyl-CoA. , 1992, Trends in biochemical sciences.

[15]  H. Willard,et al.  Purification and properties of methylmalonyl coenzyme A mutase from human liver. , 1982, Archives of biochemistry and biophysics.

[16]  M. Lidstrom,et al.  MeaB Is a Component of the Methylmalonyl-CoA Mutase Complex Required for Protection of the Enzyme from Inactivation* , 2004, Journal of Biological Chemistry.

[17]  G. Fuchs.  Alternative pathways of carbon dioxide fixation: insights into the early evolution of life? , 2011, Annual review of microbiology.

[18]  John A. Robinson,et al.  Cloning and Sequencing of the Coenzyme B12-binding Domain of Isobutyryl-CoA Mutase from Streptomyces cinnamonensis, Reconstitution of Mutase Activity, and Characterization of the Recombinant Enzyme Produced inEscherichia coli * , 1999, The Journal of Biological Chemistry.

[19]  S. Shigeoka,et al.  Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z , 2010, Archives of Microbiology.

[20]  R. Kelly,et al.  Physiological Versatility of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Supported by Transcriptomic Analysis of Heterotrophic, Autotrophic, and Mixotrophic Growth , 2009, Applied and Environmental Microbiology.

[21]  G. Fuchs,et al.  A 3-Hydroxypropionate/4-Hydroxybutyrate Autotrophic Carbon Dioxide Assimilation Pathway in Archaea , 2007, Science.

[22]  E. Miyamoto,et al.  Purification and characterization of methylmalonyl-CoA mutase from a methanol-utilizing bacterium, Methylobacterium extorquens NR-1. , 2002, Journal of nutritional science and vitaminology.

[23]  M. Lidstrom,et al.  Glyoxylate Regeneration Pathway in the Methylotroph Methylobacterium extorquens AM1 , 2002, Journal of bacteriology.

[24]  J. Rétey,et al.  Studies on methylmalonyl-CoA mutase from Propionibacterium shermanii. , 1974, European journal of biochemistry.

[25]  A. Crane,et al.  Primary structure and activity of mouse methylmalonyl-CoA mutase. , 1990, The Biochemical journal.

[26]  Hiroyuki Ogata,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 1999, Nucleic Acids Res..

[27]  G. Fuchs,et al.  3-Hydroxypropionyl-Coenzyme A Synthetase from Metallosphaera sedula, an Enzyme Involved in Autotrophic CO2 Fixation , 2007, Journal of bacteriology.

[28]  R. Kelly,et al.  Impact of Molecular Hydrogen on Chalcopyrite Bioleaching by the Extremely Thermoacidophilic Archaeon Metallosphaera sedula , 2010, Applied and Environmental Microbiology.

[29]  P. Blum,et al.  The Genome Sequence of the Metal-Mobilizing, Extremely Thermoacidophilic Archaeon Metallosphaera sedula Provides Insights into Bioleaching-Associated Metabolism , 2007, Applied and Environmental Microbiology.

[30]  R. Mackie,et al.  Comparative Analyses of Two Thermophilic Enzymes Exhibiting both β-1,4 Mannosidic and β-1,4 Glucosidic Cleavage Activities from Caldanaerobius polysaccharolyticus , 2010, Journal of bacteriology.

[31]  T. Bobik,et al.  HPLC assay for methylmalonyl–CoA epimerase , 2003, Analytical and bioanalytical chemistry.

[32]  Ruma Banerjee,et al.  The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. , 2003, Annual review of biochemistry.

[33]  W. Eisenreich,et al.  Labeling and Enzyme Studies of the Central Carbon Metabolism in Metallosphaera sedula , 2010, Journal of bacteriology.

[34]  P. Leadlay.  Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii. , 1981, The Biochemical journal.

[35]  D. E. Holloway,et al.  Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum Homologies with other cobalamin‐dependent enzymes , 1992, FEBS letters.

[36]  P. Leadlay,et al.  Subunit interactions in Propionibacterium shermanii methylmalonyl-CoA mutase studied by analytical ultracentrifugation. , 1989, The Biochemical journal.

[37]  P. Leadlay,et al.  Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. , 1989, The Biochemical journal.

[38]  G. Fuchs,et al.  Autotrophic carbon fixation in archaea , 2010, Nature Reviews Microbiology.

[39]  T. Bobik,et al.  Purification and partial characterization of the Pyrococcus horikoshii methylmalonyl-CoA epimerase , 2004, Applied Microbiology and Biotechnology.

[40]  R. Banerjee,et al.  Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase* , 1999, The Journal of Biological Chemistry.

[41]  D. E. Holloway,et al.  Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme. , 1994, The Journal of biological chemistry.

[42]  E N Baker,et al.  Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold. , 2001, Structure.

[43]  E. Miyamoto,et al.  Purification and characterization of methylmalonyl-CoA mutase from a photosynthetic coccolithophorid alga, Pleurochrysis carterae. , 2004, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[44]  R. Stjernholm,et al.  PURIFICATION AND PROPERTIES OF ENZYMES INVOLVED IN THE PROPIONIC ACID FERMENTATION , 1964, Journal of bacteriology.

[45]  R. Milo,et al.  Design and analysis of synthetic carbon fixation pathways , 2010, Proceedings of the National Academy of Sciences.

[46]  M. Adams,et al.  Extremely Thermophilic Routes to Microbial Electrofuels , 2011 .

[47]  Trevor C. Charles,et al.  Purification and characterization of homodimeric methylmalonyl-CoA mutase from Sinorhizobium meliloti , 2003, Archives of Microbiology.

[48]  H. Baker,et al.  Expression, crystallization and preliminary characterization of methylmalonyl coenzyme A epimerase from Propionibacterium shermanii. , 2001, Acta crystallographica. Section D, Biological crystallography.

[49]  P. Leadlay,et al.  How coenzyme B12 radicals are generated: the crystal structure of methylmalonyl-coenzyme A mutase at 2 A resolution. , 1996, Structure.

[50]  P. Leadlay,et al.  Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Active holoenzyme produced from Escherichia coli. , 1990, The Biochemical journal.

[51]  山田 良平.  Methylmalonyl CoA mutaseのアポ酵素異常について , 1978 .

[52]  Susumu Goto,et al.  KEGG: Kyoto Encyclopedia of Genes and Genomes , 2000, Nucleic Acids Res..

[53]  E. Liebau,et al.  Functional analysis of the methylmalonyl‐CoA epimerase from Caenorhabditis elegans , 2005, The FEBS journal.

[54]  Georg Fuchs,et al.  3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase, Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales , 2009, Journal of bacteriology.

[55]  A. Leiser,et al.  Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis , 1993, Journal of bacteriology.