Abstract
We were able to reconstitute molybdopterin (MPT)-free sulfite oxidase in vitro with the molybdenum cofactor (Moco) synthesized de novo from precursor Z and molybdate. MPT-free human sulfite oxidase apoprotein was obtained by heterologous expression in an Escherichia coli mutant with a defect in the early steps of MPT biosynthesis. In vitro reconstitution of the purified apoprotein was achieved using an incubation mixture containing purified precursor Z, purified MPT synthase, and sodium molybdate. In vitro synthesized MPT generated from precursor Z by MPT synthase remains bound to the synthase. Surprisingly, MPT synthase was found capable of donating bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT synthase when either bovine serum albumin or Moco-containing sulfite oxidase was used in place of aposulfite oxidase. After the inclusion of sodium molybdate in the reconstitution mixture, active sulfite oxidase was obtained, revealing that in vitro MPT synthase and aposulfite oxidase are sufficient for the insertion of MPT into sulfite oxidase and the conversion of MPT into Moco in the presence of high concentrations of molybdate. The conversion of MPT into Moco by molybdate chelation apparently occurs concomitantly with the insertion of MPT into sulfite oxidase.
Highlights
Fite oxidase, which can be caused by either a mutation in its structural gene or a defect in the synthesis of molybdenum cofactor (Moco), results in neurological abnormalities and often leads to death at an early age [2]
In agreement with the results obtained by Pitterle et al [6], MPT formed by incubation of active MPT synthase with precursor Z remains bound to MPT synthase (Fig. 2)
high-performance liquid chromatography (HPLC) analysis revealed that form A could be obtained from active MPT synthase (Fig. 2A), whereas no form A was obtained from inactive MPT synthase (Fig. 2B)
Summary
Molybdenum cofactor; MPT, molybdopterin; HPLC, high-performance liquid chromatography; BSA, bovine serum albumin; Ni-NTA, nickel-nitrilotriacetic acid. The reactions of the Moco biosynthetic pathway comprise three stages that are identical in all organisms using molybdoenzymes: (i) conversion of a guanine nucleotide into the meta-stable precursor Z, (ii) conversion of precursor Z into MPT, and (iii) insertion of molybdenum into MPT, forming Moco. In the activated form of MPT synthase, the C terminus of the small subunit is converted to a glycine thiocarboxylate that acts as the sulfur donor for the conversion of precursor Z to MPT. We were able to reconstitute sulfite oxidase activity by the inclusion of molybdate in the in vitro system These results reveal that no other proteins are required for the in vitro insertion of MPT into sulfite oxidase or for the conversion of MPT into Moco under the conditions used in the reconstitution mixture
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