Functionality of alternative splice forms of the first enzymes involved in human molybdenum cofactor biosynthesis.
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 (mocs1 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 MOCS1A, but without the double glycine motif, and the entire MOCS1B. Here we describe the first functional characterization of the human proteins MOCS1A and MOCS1B as well as the MOCS1A-MOCS1B 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 MOCS1A and MOCS1B in Escherichia coli resulted in the formation and accumulation of precursor Z that was subsequently converted to MPT. We showed that for catalytic activity MOCS1A needs an accessible C-terminal double glycine motif. In the MOCS1A-MOCS1B fusion proteins lacking the MOCS1A double glycines, only MOCS1B activity could be detected. No evidence was found for an expression of MOCS1B from the bicistronic mocs1A-mocs1B splice type I cDNA, indicating that MOCS1B is only expressed as a fusion to an inactive MOCS1A. Comparative mutational studies of MOCS1A and the small subunit of the E. coli MPT synthase (MoaD) indicate a different function of the double glycine motifs in both proteins.
Hänzelmann
Petra
P
Institute of Plant Biology, Technical University of Braunschweig, D-38023 Braunschweig, Germany.
Schwarz
Gunter
G
Mendel
Ralf R
RR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D019755:Carbon-Carbon Lyases; D003067:Coenzymes; D017931:DNA Primers; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008667:Metalloproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D011621:Pteridines; D017386:Sequence Homology, Amino Acid; D013466:Sulfurtransferases / Q000737:chemistry / Q000235:genetics
D019755:Carbon-Carbon Lyases; D003067:Coenzymes; D017931:DNA Primers; D005998:Glycine; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009687:Nuclear Proteins; D011621:Pteridines; D013466:Sulfurtransferases; C022261:molybdopterin; C085585:molybdopterin synthase; C114840:MOCS1 protein, human