Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii

Biochemistry. 2009 Jun 30;48(25):5882-9. doi: 10.1021/bi8023114.

Abstract

The archaeal cobY gene encodes the nonorthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62)/GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbl (coenzyme B(12)). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein (Mj CobY) that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (K(d) = 5 muM), but it does not bind the GTP analogues GMP-PNP (guanosine 5'-(beta,gamma)-imidotriphosphate) or GMP-PCP (guanylyl 5'-(beta,gamma)-methylenediphosphonate) nor GDP. Results from ITC experiments indicate that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5'-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for AdoCbi-P (K(d) = 0.76 muM) but did not bind nonadenosylated Cbi-P. Kinetic parameters for theMj CobY reaction were determined. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80 degrees C with a concomitant loss of activity. We propose that ORF MJ1117 of M. jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Archaeal Proteins / biosynthesis
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Guanosine Triphosphate / chemistry*
  • Guanosine Triphosphate / metabolism
  • Methanococcales / enzymology*
  • Methanococcales / genetics
  • Multienzyme Complexes / biosynthesis
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Nucleotidyltransferases / biosynthesis
  • Nucleotidyltransferases / chemistry*
  • Nucleotidyltransferases / genetics
  • Pentosyltransferases / biosynthesis
  • Pentosyltransferases / chemistry*
  • Pentosyltransferases / genetics
  • Protein Binding / genetics
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics

Substances

  • Archaeal Proteins
  • Multienzyme Complexes
  • Recombinant Proteins
  • Guanosine Triphosphate
  • Pentosyltransferases
  • nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase
  • Nucleotidyltransferases