The biosynthesis of molybdopterin in Escherichia coli. Purification and characterization of the converting factor.
Molybdopterin, the universal component of the pterin molybdenum cofactors, contains a dithiolene group serving to bind Mo. Addition of the dithiolene sulfurs to a molybdopterin precursor requires the activity of the converting factor. Active converting factor has been purified from Escherichia coli chlA1 cells and found to have two subunits of mass 10 and 16 kDa. Electrophoresis of the purified converting factor on denaturing polyacrylamide gels revealed the presence of a 27-kDa protein as well. Partial NH2-terminal amino acid sequencing showed that the 27-kDa protein is a covalent complex of the 10- and 16-kDa proteins. The inactive converting factor purified from E. coli chlN contains both subunits, as established by amino acid sequencing of the purified material, but the 10-kDa subunit is inactive. Absence of the 27-kDa complex in chlN preparations showed that the inactive covalent complex is only formed from the active converting factor. Evidence that activation of the small subunit requires the acquisition of sulfur was obtained from the difference in the molecular masses of the 10-kDa proteins isolated from chlA1 and chlN cells and from the differential sensitivities of the chlA1 and chlN converting factors to iodoacetamide.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
D M
DM
Pitterle
K V
KV
Rajagopalan
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003067:Coenzymes / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D007460:Iodoacetamide / Q000737:chemistry; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D011621:Pteridines / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D007460:Iodoacetamide; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D011621:Pteridines; D013466:Sulfurtransferases; C022261:molybdopterin; C085585:molybdopterin synthase