3rsb | pdb_00003rsb
Structure of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii
Structural highlights
FunctionCOBY_METJA Guanylyltransferase that catalyzes the synthesis of adenosylcobinamide-GDP (AdoCbi-GDP) from adenosylcobinamide-phosphate (AdoCbi-P) and GTP. Is involved in adenosylcobalamin biosynthesis. Binds one GTP per dimer. Can not use other NTPs or GDP. Does not display AdoCbi kinase activity. Is also able to catalyze the condensation of 2-phospho-L-lactate (LP) with GTP in vitro to form PPi and (2S)-lactyl-2-diphospho-5'-guanosine (LPPG), but is much less efficient than CofC, the presumed enzyme catalyzing this reaction in vivo.[1] [2] Publication Abstract from PubMedIn archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP dependent corrinoid phosphorylation and GTP dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY)1 in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in >94% loss of catalytic activity, thus variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobYG153D had 10-fold higher affinity for GTP than MjCobTWT, but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobYG153D in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyses the formation of uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc). Structure and Mutational Analysis of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii: Insights into GTP Binding and Dimerization.,Newmister SA, Otte MM, Escalante-Semerena JC, Rayment I Biochemistry. 2011 May 4. PMID:21542645[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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