Abstract
The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t((1/2)) of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6 Angstrom resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.
Highlights
Molybdenum enzymes, molybdenum is found as molybdenum cofactor (Moco)3 [2] that consists of molybdenum covalently bound to the dithiolate moiety of a tricyclic pterin referred to as molybdopterin or metal-binding pterin (MPT), whose structure is conserved in eukaryotes, eubacteria, and archaea [3]
Ent E. coli strains that are free of MPT (BL21) [10] or accumulate MPT (RK5206) [36], Moco, or Wco (TP1000) [37], the latter one depending on the addition of molybdate or tungstate to the media
Its total pterin content was determined by HPLC FormA analysis, a method that cannot discriminate between MPT and Moco/Wco
Summary
Molybdenum cofactor; Wco, tungstensubstituted form of Moco; MCP, Moco carrier protein; MPT, molybdopterin or metal-binding pterin; NR, nitrate reductase; r.m.s.d., root mean square deviation; Se-Met, seleno-methionated; SO, sulfite oxidase; HPLC, high performance liquid chromatography. MCP activity was measured with the nit-1 reconstitution assay [19] without any denaturing procedure, indicating that bound Moco is transferred to apo-nitrate reductase (apo-NR) It is unknown whether MCP is able to donate Moco to molybdenum enzymes other than NR. Clustering of conserved and mobile surface residues together with in silico docking studies depicted a surface cavity as a putative Moco-binding site, which was supported by structure-guided mutagenesis and functional studies
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