Complex structure of NADPH-cytochrome P450 reductase and heme oxygenase-1

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to FAD in CPR, then to FMN in CPR, and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, ``closed-open transition'' of CPR is indispensable because FMN in the closed conformation of CPR is covered by FAD-binding domain, thus FMN is not exposed to the surface in the closed conformation. Recently, Hamdane et al. determined the crystal structures of a hinge-shortened rat CPR variant (ΔTGEE), which favors an open conformation [1]. In the open conformation of CPR, FMN is exposed to the surface, thus this conformation appears to be favorable to interact with the redox partners, though no complex structure of CPR and its redox partner has been determined. Here, we demonstrate that ΔTGEE makes a stable complex with heme-rat HO-1 (rHO-1) complex and can support HO reaction, though its efficiency is extremely limited. Further we determine the crystal structure of ΔTGEE in complex with heme-rHO-1 at 4.3 Å resolution [2]. X-ray scattering and biochemical data suggest that the complex structure of ΔTGEE and heme-rHO-1 is similar to that of wild type CPR and heme-rHO-1. Distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme. On the other hand, FAD is far from FMN and heme, indicating that the ``closed-open transition'' of CPR is required for electron transfer from FAD to FMN.

Crystal structures of γ-glutamyltranspeptidase in complex with inhibitors

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is involved in the degradation of γ-glutamyl compounds such as glutathione (GSH; γ-glutamyl-cysteinyl-glycine) . A major physiological role of this enzyme is to cleave the extracellular GSH as a source of cysteine for intracellular glutathione biosynthesis. Another crucial role of GGT is to cleave glutathione-S-conjugates as a key step in detoxification of xenobiotics and drug metabolism. In mammals, GGT has been implicated in physiological disorders such as Parkinson's disease, other neurodegenerative diseases including Alzheimer's disease and cardiovascular disease. The indispensable roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress suggest that GGT is an attractive pharmaceutical target. We here report the binding mode of acivicin, a well-known glutamine antagonist, to B. subtilis GGT at 1.8 Å resolution showing that acivicin is bound to the Oγ atom of Thr403, the catalytic nucleophile of the enzyme, through its C3 atom [1]. The observed electron density around the C3 atom was best fitted to the planar and sp2 hybridized carbon, consistent with a simple nucleophilic substitution of Cl at the imino carbon by Oγ atom of Thr403. Furthermore, comparison of three bacterial enzymes, the GGTs from E. coli, H. pylori and B. subtilis in complex with acivicin, showed significant diversity in the orientation of the dihydroisoxazole ring among three GGTs. The differences are discussed in terms of the recognition of the α-amino and α-carboxy groups in preference to the dihydroisoxazole ring as observed in time-lapse soaking crystal structures of B. subtilis GGT with acivicin.

Neutron Structure of Phycocyanobilin:oxidoreductase Complexed with Biliverdin

Phytobilins are linear tetrapyrrole compounds used as chromophore for light harvesting and photoreceptor proteins in higher plants, algae, and cyanobacteria. Phytobilins are synthesized from biliverdin IX(alpha) (BV). Phycocianobilin:oxidoreductase (PcyA) is an enzyme to produce phycocyanobilin (PCB) used as chromophore for light harvesting and photoreceptor proteins. PcyA is unique because it catalyzes the reduction of BV by two sequential steps; the first step is the reduction of the vinyl of the BV D-ring to produce 18(1)-18(2)-dihydrobiliverdin (18EtBV), and the second step is the reduction of the A-ring. In these reduction steps, four hydrogen atoms are delivered to BV. The earlier studies showed that the carboxyl group of Asp105 showed dual conformations. This has been attributed to the difference of its protonation states. The catalytically essential His88 was suggested to be protonated (i. e. His88 is a proton donor) to donate the proton to BV. BVH+ (N-protonated) state, in which four pyrrole N atoms of BV were fully protonated, was proposed to be partially formed when BV was bound to PcyA. Further, another tautomeric BVH+ state in which three of four pyrrole N atoms of BV were protonated and the lactam (C=O) group of BV D-ring was protonated as lactim (C-OH; O-protonated) was proposed. Additionally, newly identified water molecule near BV has been suggested to be a proton donor. To elucidate the H atom positions of these molecules, we determined the neutron crystal structure of the PcyA-BV complex at 1.95 Å resolution. Crystal with approximately 2.2 X 1.8 X 0.8 mm3 size, which was soaked into the deuterium-exchanged crystallization solution, was used in the diffraction experiment. The neutron diffraction intensity data was collected using IBARAKI Biological Crystal Diffractometer (iBIX) in J-PARC. In this conference, we report the protonation states of catalytically important residues and BV as well as orientations of water molecules in the PcyA-BV complex.

Structural analyses of the two cysteine desulfurases from Aquifex aeolicus

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer to a wide range of acceptor proteins. IscS degrades L-cysteine into L-alanine and a sulfur atom in a pyridoxal 5'-phosphate (PLP) dependent manner. In this reaction, it is essential for a conserved Lys residue of IscS to form Schiff base (the covalent bonding interaction) with PLP. Recent accumulations of genomic information have revealed that some IscS homologues in archaea and thermophilic bacteria lack this invariant Lys. Here we report the crystal structures of two paralogous cysteine desulfurases, the canonical Aa IscS1 and the invariant Lys lacking Aa IscS2, from Aquifex aeolicus. Aa IscS1/Aa IscS2 were overproduced in E. coli, and purified by heat-treatment and several column chromatography, and crystallized. The structure of Aa IscS1 was determined at 2.00 Å (Rcryst= 19.4% and Rfree = 22.0%), and Aa IscS2 at 2.55 Å (Rcryst= 21.8% and Rfree = 27.0%). Overall structures as well as orientations of the residues in the active site were quite similar to each other. In Aa IscS1 the PLP adduct was anchored in the catalytic pocket of Aa IscS1 by the formation of the aldimine Schiff base with the invariant Lys. Whereas in Aa IscS2 the PLP was not seen in the active pocket, since the catalytic Lys was substituted by Leu. Alternatively, an electron density derived from unknown-small molecule was located in the catalytic site of Aa IscS2. The shape of this electron density was completely different from that of PLP. The Bijvoet difference map calculated from data collected at λ=1.7 Å overlapped with the electron density observed in the active site; the unknown-small molecule probably contains such metals as iron atoms. Furthermore, the ICP-MS analysis demonstrated that as-isolated Aa IscS2 harbored the iron atom in the solution state. More recently we obtained the experimental evidences that non-canonical Aa IscS2 was able to form the binary complex with Aa IscU, which is responsible for a scaffold for the assembly of a nascent Fe-S cluster. Base on the structural/biochemcal results, possible physiological functions of two cysteine desulfrurases will be discussed.