No abstract

In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].

We have examined the rapid reaction kinetics and spectroscopicpropertiesofthemolybdenum-containing,NAD ؉ -dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo V state and demonstrated the direct transfer of the substrate C␣ hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.The molybdenum-containing, NAD ϩ -dependent formate dehydrogenases from bacteria such as Ralstonia eutropha catalyze the oxidation of formate to CO 2 , reducing NAD ϩ to NADH, and are members of the NADH dehydrogenase superfamily of enzymes. These cytosolic formate dehydrogenases are expressed under aerobic conditions and are distinct from the predominant bacterial formate dehydrogenases expressed under anaerobic conditions, which are typically membrane-associated and extremely O 2 -sensitive; they are also distinct from the cofactorless formate dehydrogenases from many eukaryotes, including humans, that catalyze the direct hydride transfer from formate to NAD ϩ (1). In R. eutropha, the trimeric FdsABG (soluble, NAD ϩ -dependent, molybdenum-containing formate dehydrogenase, product of the fdsABG gene cluster) enzyme is encoded by the fdsGBACD operon and is predicted to contain seven iron/sulfur clusters, FMN, and a molybdenum center; each subunit bears significant sequence similarity (ϳ21% identity) and expected strong structural homology to a corresponding subunit of the matrix-or cytosol-exposed portion of NADH dehydrogenase, and the spatial layout of the several redox-active centers is particularly highly conserved. The 105-kDa FdsA is cognate to subunit Nqo3 in the crystallographically characterized Thermus thermophilus NADH dehydrogenase (2, 3) and is predicted to have four [4Fe-4S] clusters and one [2Fe-2S] cluster (4). The close structural homology predicted between FdsA and Nqo3 extends to the presence of a histidine ligand to one of the [4Fe-4S] clusters near the N terminus of the FdsA/Nqo3 subunit. The C terminus of FdsA contains a molybdenum center and has ϳ60% sequence similarity to the structurally characterized molybdenum-containing formate dehydrogenase FdhF of Escherichia ...

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b 5 , and NADH/FADdependent cytochrome b 5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactorbinding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/ MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo-or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.In eukaryotes the trace element molybdenum is essential for a number of enzymes where the molybdenum atom is part of the so-called molybdenum cofactor (Moco) 2 in the active site of these enzymes (1). Moco is a pterin-based cofactor with a C6-substituted pyrano ring, a terminal phosphate, and a unique dithiolate group that binds the molybdenum atom. Moco-containing enzymes (Mo-enzymes) catalyze important reactions in the global carbon, sulfur, and nitrogen cycles that are characterized by transfer of an oxygen atom to or from a substrate. In mammals, one Mo-enzyme is sulfite oxidase (SO), which catalyzes the last step in the degradation of sulfur-containing amino acids and sulfatides (2). The active SO protein is a homodimer with each monomer of ϳ52 kDa consisting of a N-terminal cytochrome b 5 (cyt b 5 )/heme-binding domain and a C-terminal Moco-binding domain, the latter also harboring the dimerization interface. Both the Moco-and the heme-binding domain of mammalian SO are similar to the respective domains of nitrate reductase (NR), which catalyzes the first and rate-limiting step in nitrate assimilation in autotrophic organisms like plants, algae, and fungi (3). In addition to its N-terminal Mocobinding domain and the cytb 5 /heme-binding domain, each NR monomer possesses a C-terminal FAD-binding domain. Xanthine oxidoreductase (XOR) is another mammalian Mo-enzyme, and it is active as a homodimer with each ϳ145-kDa monomer consisting of several distinct domains: an N-terminal domain...