Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica.
In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT(WT)) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT(WT). The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.
Claas
Kathy R
KR
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Parrish
J R
JR
Maggio-Hall
L A
LA
Escalante-Semerena
J C
JC
D000483:Alleles; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001562:Benzimidazoles / Q000378:metabolism*; D015153:Blotting, Western; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D002876:Chromosomes, Bacterial / Q000235:genetics; D003038:Cobamides / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D001562:Benzimidazoles; D003038:Cobamides; D009537:Nicotinamide Mononucleotide; C002953:nicotinate mononucleotide; C015158:5,6-dimethylbenzimidazole; D010430:Pentosyltransferases; C000913:cobamamide