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

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.

• Authors: Michele M Otte, Jorge C Escalante-Semerena
• Journal: Biochemistry (Biochemistry) Vol. 48 Issue 25 Pg. 5882-9 (Jun 30 2009) ISSN: 1520-4995 [Electronic] United States
• PMID: 19489548 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural)

Chemical References

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

Topics

• Archaeal Proteins (biosynthesis, chemistry, genetics)
• Guanosine Triphosphate (chemistry, metabolism)
• Methanococcales (enzymology, genetics)
• Multienzyme Complexes (biosynthesis, chemistry, genetics)
• Nucleotidyltransferases (biosynthesis, chemistry, genetics)
• Pentosyltransferases (biosynthesis, chemistry, genetics)
• Protein Binding (genetics)
• Recombinant Proteins (biosynthesis, chemistry, genetics)