Abstract
Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans. Genetic deficiencies of enzymes involved in cofactor biosynthesis in humans lead to a severe and usually fatal disease. The molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin, that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of molybdopterin is generated by molybdopterin synthase, which consists of a large (MoaE) and small (MoaD) subunit. The crystal structure of molybdopterin synthase revealed a heterotetrameric enzyme in which the C terminus of each MoaD subunit is deeply inserted into a MoaE subunit to form the active site. In the activated form of the enzyme, the MoaD C terminus is present as a thiocarboxylate. The present study identified the position of the thiocarboxylate sulfur by exploiting the anomalous signal originating from the sulfur atom. The structure of molybdopterin synthase in a novel crystal form revealed a binding pocket for the terminal phosphate of molybdopterin, the product of the enzyme, and suggested a binding site for the pterin moiety present in precursor Z and molybdopterin. Finally, the crystal structure of the MoaE homodimer provides insights into the conformational changes accompanying binding of the MoaD subunit.
Highlights
Molybdenum cofactor biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea, and eukaryotes, including humans
Localization of the Sulfur Atom in the MoaD Thiocarboxylate—To locate the sulfur atom in the MoaD thiocarboxylate, activated MPT synthase was crystallized in the monoclinic crystal form, and diffraction data were collected to a resolution of 2.5 Å utilizing x-rays with a wavelength of 1.5418 Å (CuK␣) to improve the anomalous signal originating from sulfur atoms
MPT synthase catalyzes the conversion of precursor Z into MPT through sulfur transfer from the MoaD thiocarboxylate
Summary
Molybdenum cofactor; MPT, molybdopterin; r.m.s., root mean square; E1, ubiquitin-activating enzyme. In order for MoaD to interact with both MoeB and MoaE, MoaD must reversibly bind to either protein (Fig. 1a) This complex interaction is vital for the action of MPT synthase, where two MoaD subunits transfer the required sulfur atoms in two subsequent steps [12]. While the majority of these residues originate from the proximal MoaE subunit, two residues (His103Ј and Arg-104Ј) are located in the distal MoaE subunit Based on their phylogenetic conservation and their location in close proximity to the active site, these residues have been postulated to be important for the function of MPT synthase [13]. We present here the 1.9-Å resolution crystal structure of MPT synthase in a novel orthorhombic space group This structure revealed a bound sulfate ion near the MoaD C terminus that probably mimics the binding site for the terminal phosphate group of MPT, the product of the reaction. Taken together with these data, the structures presented here further define the mechanism of MPT synthase and the role of specific residues in its catalytic mechanism
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
