In vitro synthesis of molybdopterin from precursor Z using purified converting factor. Role of protein-bound sulfur in formation of the dithiolene.
The pterin component of the molybdenum cofactor, termed molybdopterin, is synthesized in Escherichia coli by enzymes encoded at the chl loci. A late step in the biosynthetic pathway, the conversion of a molybdopterin intermediate, precursor Z, to molybdopterin, requires the activity of a two-subunit protein, the converting factor. Precursor Z has many of the features of molybdopterin but lacks the dithiolene function essential for molybdenum ligation. Conversion of precursor Z to molybdopterin is accomplished by transfer of sulfur to produce the dithiolene. The present study describes an in vitro system for molybdopterin biosynthesis comprised of purified precursor Z and purified converting factor. It is established that these components are sufficient to yield molybdopterin, identified by conversion to its characteristic products, Form A, Form B, and dicarboxamidomethylmolybdopterin. Under conditions of precursor excess, the formation of molybdopterin was stoichiometric with converting factor, as would be expected in the absence of a sulfur-regenerating system. The labile product of the reaction, molybdopterin, remained associated with the converting factor large subunit. These results establish that the source of sulfur for molybdopterin biosynthesis is the converting factor and suggest that in vivo a novel sulfur cycle must function to resupply sulfur to the converting factor.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
D M
DM
Pitterle
J L
JL
Johnson
K V
KV
Rajagopalan
D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism*
D001426:Bacterial Proteins; D003067:Coenzymes; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009943:Organophosphorus Compounds; D011498:Protein Precursors; D011621:Pteridines; D013455:Sulfur; D013466:Sulfurtransferases; C081783:1-(2-amino-4-oxopteridin-7-yl)-1-oxo-2,3,4-butanetriol-2,4-cyclic phosphate; C022261:molybdopterin; C085585:molybdopterin synthase