Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate.

George A O'Toole,Jorge C Escalante-Semerena

doi:10.1074/jbc.270.40.23560

George A O'Toole, Jorge C Escalante-Semerena

Open Access

https://doi.org/10.1074/jbc.270.40.23560

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Abstract

The CobU protein of Salmonella typhimurium was overexpressed and purified to approximately 94% homogeneity. N-terminal sequencing of purified CobU confirmed the first 22 amino acids. In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamide-phosphate intermediate. We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme-guanylyl intermediate. In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. Treatment of inactive CobU with dithiothreitol restored approximately 20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity. The sulfhydryl modifying agents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide abolished both CobU activities. Native CobU protein was a dimer (approximately 40 kDa) that functioned optimally at pH 8.8-9.0 and 37 degrees C. Substrates and kinetic parameters for both activities were determined. The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen.

Highlights

In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost
CobT catalyzes the transfer of the phosphoribosyl moiety of nicotinic acid mononucleotide to dimethylbenzimidazole to yield ␣-ribazole5Ј-phosphate [1], which is dephosphorylated by CobC to generate ␣-ribazole [2]
We report the overexpression and purification of the cobU gene product, document that the CobU protein has both kinase and guanylyltransferase activities required for the synthesis of Ado-CBI-GDP from Ado-CBI, and provide evidence that AdoCBI-P is an intermediate in the synthesis of Ado-CBI-GDP

Summary

Introduction

In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost. In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen. Of the nucleotide loop of cobalamin in Salmonella typhimurium is thought to require the involvement of four enzymes, CobU, CobS, CobT, and CobC (Fig. 1). CobT catalyzes the transfer of the phosphoribosyl moiety of nicotinic acid mononucleotide to dimethylbenzimidazole to yield ␣-ribazole5Ј-phosphate [1], which is dephosphorylated by CobC to generate ␣-ribazole [2]. We report the overexpression and purification of the cobU gene product, document that the CobU protein has both kinase and guanylyltransferase activities required for the synthesis of Ado-CBI-GDP from Ado-CBI, and provide evidence that AdoCBI-P is an intermediate in the synthesis of Ado-CBI-GDP. The characterization of homogeneous CobU protein includes an analysis of substrate specificity, physical characteristics, and kinetic parameters

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