Identification of Guanosine as the Nucleoside Moiety of the Molybdopterin Cofactor of Arsenite Oxidase

Abstract

Arsenite oxidase converts arsenite (As+3 ) to arsenate (As+s), thereby enabling bacteria that produce the enzyme to tolerate high levels of arsenite in the environment. The electron transfer occurs via a molybdenum atom liganded to a molybdopterin cofactor associated with the enzyme. Molybdopterin cofactors from all bacteria that have been investigated contain an associated nucleotide. However, the nucleotide associated with the cofactor in arsenite oxidase has not been reported. To determine which nucleotide is attached to the cofactor in arsenite oxidase, the cofactor was released from the enzyme by heat denaturation in the presence of acid. The nucleotide moiety was hydrolyzed with pyrophosphatase and alkaline phosphatase, and separated on reverse phase high performance liquid chromatography. Four nucleotide standards were treated similarly, and their retention times compared to the one released from the enzyme. The results suggest that guanosine is the nucleotide associated with the molybdopterin cofactor of arsenite oxidase.