Structure and Mutational Analysis of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii: Insights into GTP Binding and Dimerization

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• 作者：Sean A. Newmister ; Michele M. Otte ; Jorge C. Escalante-Semerena ; Ivan Rayment
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：June 14, 2011
• 年：2011
• 卷：50
• 期：23
• 页码：5301-5313
• 全文大小：1216K
• 年卷期：v.50,no.23(June 14, 2011)
• ISSN：1520-4995

文摘

In 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) 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 鈮?4% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY^G153D had 10-fold higher affinity for GTP than MjCobY^WT 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 MjCobY^G153D 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 catalyzes the formation of uridine diphosphate鈥?i>N-acetylglucosamine (UDP鈥揋lcNAc).