Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii.
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.
Otte
Michele M
MM
Department of Bacteriology, University of Wisconsin at Madison, 1550 Linden Drive, Madison, Wisconsin 53706-1521, USA.
Escalante-Semerena
Jorge C
JC
D019843:Archaeal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D017015:Methanococcales / Q000201:enzymology* / Q000235:genetics; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D010430:Pentosyltransferases; C028015:nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase; D009713:Nucleotidyltransferases