The Methanosarcina mazei MM2060 Gene Encodes a Bifunctional Kinase/Decarboxylase Enzyme Involved in Cobamide Biosynthesis

Biochemistry. 2018 Jul 31;57(30):4478-4495. doi: 10.1021/acs.biochem.8b00546. Epub 2018 Jul 13.


Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate that open reading frame MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with l-threonine- O-3-phosphate (l-Thr-P) decarboxylase (EC and l-Thr kinase activities (EC In Salmonella enterica, where Cba biosynthesis has been extensively studied, the activities mentioned above are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein ( MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and l-Thr as substrates and generated ADP, l-Thr-P, and ( R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111-amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display activity either in vivo or in vitro. Although the C-terminal MmCobD domain was not required for l-Thr-P decarboxylase or l-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had a 1000-fold higher affinity for ATP and a catalytic efficiency 1300-fold higher than that of MmCobD purified under oxic conditions. To the best of our knowledge, MmCobD is the first example of a new class of l-Thr-P decarboxylases that also have l-Thr kinase activity. An archaeal protein with l-Thr kinase activity had not been identified prior to this work.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Biosynthetic Pathways*
  • Carboxy-Lyases / chemistry
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism*
  • Cobamides / genetics
  • Cobamides / metabolism*
  • Methanosarcina / chemistry
  • Methanosarcina / genetics
  • Methanosarcina / metabolism*
  • Open Reading Frames
  • Protein Kinases / chemistry
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Sequence Alignment
  • Substrate Specificity


  • Archaeal Proteins
  • Cobamides
  • Adenosine Triphosphate
  • Protein Kinases
  • Carboxy-Lyases