Analysis of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2. Identification of residue His-46 as the site of guanylylation.
CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B(12)) biosynthesis in Salmonella typhimurium LT2. In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis. The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate. Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2'-OH group of the ribose, being important for this recognition. During the kinase reaction, both the nucleotide base and the 2'-OH group of the ribose are important for gamma-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction. Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation. Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP. Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation.
Thomas
M G
MG
Departments of Bacteriology and Biochemistry and Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706, USA.
Thompson
T B
TB
Rayment
I
I
Escalante-Semerena
J C
JC
D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003038:Cobamides / Q000096:biosynthesis; D006639:Histidine; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity
D003038:Cobamides; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C414173:adensoylcobinamide; D006639:Histidine