Functionality of alternative splice forms of the first enzymes involved in human molybdenum cofactor biosynthesis

Abstract

In humans, genetic deficiencies of enzymes involved in molybdenum cofactor biosynthesis trigger an autosomal recessive and usually fatal disease with severe mostly neurological symptoms. In each of the three biosynthesis steps, at least two proteins or domains are linked for catalysis. For steps 1 and 2, bicistronic mocs (molybdenum cofactor synthesis) mRNAs were found (mocsl and mocs2) that have been proposed to encode two separate proteins (A and B). In both cases, the A proteins share a highly conserved ubiquitin-like double glycine motif, which is functionally important at least for the small subunit of molybdopterin (MPT) synthase (MOCS2A). Besides the bicistronic form of mocs1, two alternative splice transcripts were found, resulting in the expression of multidomain proteins embodying both MOCSIA, but without the double glycine motif, and the entire MOCSIB. Here we describe the first functional characterization of the human proteins MOCSIA and MOCSIB as well as the MOCSIA-MOCSIB fusion proteins that catalyze the formation of precursor Z, a 6-alkyl pterin with a cyclic phosphate, the immediate precursor of MPT in molybdenum cofactor biosynthesis. High level expression of MOCSIA and MOCSIB in Escherichia coli resulted in the formation and accumulation of precursor Z that was subsequently converted to MPT. We showed that for catalytic activity MOCSIA needs an accessible C-terminal double glycine motif. In the MOCSIA-MOCSIB fusion proteins lacking the MOCSIA double glycines, only MOCSIB activity could be detected. No evidence was found for an expression of MOCSIB from the bicistronic mocs1A-mocs1B splice type I cDNA, indicating that MOCSIB is only expressed as a fusion to an inactive MOCSIA. Comparative mutational studies of MOCSIA and the small subunit of the E. coli MPT synthase (MoaD) indicate a different function of the double glycine motifs in both proteins.