Hiroshi Sugimoto

Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase

Dissecting Nitric Oxide Reductase Bacterial breakdown of nitrogen compounds in soil and the oceans provides the largest emission source of the greenhouse gas, nitrous oxide (N2O). A key enzyme in this process is nitric oxide reductase (NOR), which catalyzes the reduction of nitric oxide (NO) to N2O. Hino et al. (p. 1666, published online 25 November; see the Perspective by Moënne-Loccoz and Fee) now describe the crystal structure of NOR from Pseudomonas aeruginosa. Consistent with their evolutionary relatedness, the transmembrane region topology and arrangement of metal centers in NOR are similar to those in cytochrome oxidases, key enzymes in aerobic respiration. A structural comparison gives insight into the features that allow conversion between nitric oxide and oxygen reduction. Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N2O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (FeB) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

Crystal structure of the macrophage migration inhibitory factor from rat liver

The tertiary structure of the macrophage migration inhibitory factor (MIF) from rat liver (12,300 Mr) is presented at 2.2 Å resolution. Each monomer consists of two β/α/β motifs aligned in quasi two-fold symmetry, comprising a domain consisting of a four-stranded mixed β-sheet and two antiparallel α-helices. The protein exists as a trimer in the crystal. An extra β-strand that is almost perpendicular to the other β-strands joins to the β-sheet of the neighbouring monomer in the trimer. Unexpected similarities were detected between MIF and two kinds of isomerase.

Determination of damage-free crystal structure of an X-ray–sensitive protein using an XFEL

We report a method of femtosecond crystallography for solving radiation damage–free crystal structures of large proteins at sub-angstrom spatial resolution, using a large single crystal and the femtosecond pulses of an X-ray free-electron laser (XFEL). We demonstrated the performance of the method by determining a 1.9-Å radiation damage–free structure of bovine cytochrome c oxidase, a large (420-kDa), highly radiation-sensitive membrane protein.

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Staurosporine isolated from Streptomyces sp. TP-A0274 is a member of the family of indolocarbazole alkaloids that exhibit strong antitumor activity. A key step in staurosporine biosynthesis is the formation of the indolocarbazole core by intramolecular C–C bond formation and oxidative decarboxylation of chromopyrrolic acid (CPA) catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we report x-ray crystal structures of CPA-bound and -free forms of StaP. Upon substrate binding, StaP adopts a more ordered conformation, and conformational rearrangements of residues in the active site are also observed. Hydrogen-bonding interactions of two carboxyl groups and T-shaped π–π interactions with indole rings hold the substrate in the substrate-binding cavity with a conformation perpendicular to the heme plane. Based on the crystal structure of StaP–CPA complex, we propose that C–C bond formation occurs through an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I [Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738–740]. The subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.

Structure of PAS-linked histidine kinase and the response regulator complex

We determined the structure of the complex of the sensory histidine kinase (HK) and its cognate response regulator (RR) in the two-component signal transduction system of Thermotoga maritima. This was accomplished by fitting the high-resolution structures of the isolated HK domains and the RR onto the electron density map (3.8 A resolution) of the HK/RR complex crystal. Based on the structural information, we evaluated the roles of both interdomain and intermolecular interactions in the signal transduction of the cytosolic PAS-linked HK and RR system, in particular the O(2)-sensor FixL/FixJ system. The PAS-sensor domain of HK interacts with the catalytic domain of the same polypeptide chain by creating an interdomain beta sheet. The interaction site between HK and RR, which was confirmed by NMR, is suitable for the intermolecular transfer reaction of the phosphoryl group, indicating that the observed interaction is important for the phosphatase activity of HK that dephosphorylates phospho-RR.

Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus

The structure of quinol-dependent nitric oxide reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N2O, has been characterized at 2.5 Å resolution. The overall fold of qNOR is similar to that of cytochrome c–dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

Density Functional Theory Study on a Missing Piece in Understanding of Heme Chemistry : The Reaction Mechanism for Indoleamine 2,3-Dioxygenase and Tryptophan 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing dioxygenases and catalyze oxidative cleavage of the pyrrole ring of L-tryptophan. On the basis of three recent crystal structures of these heme-containing dioxygenases, two new mechanistic pathways were proposed by several groups. Both pathways start with electrophilic addition of the Fe(II)-bound dioxygen concerted with proton transfer (oxygen ene-type reaction), followed by either formation of a dioxetane intermediate or Criegee-type rearrangement. However, density functional theory (DFT) calculations do not support the proposed concerted oxygen ene-type and Criegee-type rearrangement pathways. On the basis of DFT calculations, we propose a new mechanism for dioxygen activation in these heme systems. The mechanism involves (a) direct electrophilic addition of the Fe(II)-bound oxygen to the C2 or C3 position of the indole in a closed-shell singlet state or (b) direct radical addition of the Fe(III)-superoxide to the C2 position of the indole in a triplet (or open-shell singlet) state. Then, a radical-recombination or nearly barrierless charge-recombination step from the resultant diradical or zwitterionic intermediates, respectively, proceeds to afford metastable dioxetane intermediates, followed by ring-opening of the dioxetanes. Alternatively, homolytic O-O bond cleavage from the diradical intermediate followed by oxo attack and facile C2-C3 bond cleavage could compete with the dioxetane formation pathway. Effects of ionization of the imidazole and negatively charged oxyporphyrin complex on the key dioxygen activation process are also studied.

ONIOM Study on a Missing Piece in Our Understanding of Heme Chemistry: Bacterial Tryptophan 2,3-Dioxygenase with Dual Oxidants

Unique heme-containing tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze oxidative cleavage of the pyrrole ring of L-tryptophan (Trp). Although these two heme dioxygenases were discovered more than 40 years ago, their reaction mechanisms were still poorly understood. Encouraged by recent X-ray crystal structures, new mechanistic pathways were proposed. We performed ONIOM(B3LYP:Amber) calculations with explicit consideration of the protein environment to study various possible reaction mechanisms for bacterial TDO. The ONIOM calculations do not support the proposed mechanisms (via either formation of the dioxetane intermediate or Criegee-type rearrangement); a mechanism that is exceptional in the hemes emerges. It starts with (1) direct radical addition of a ferric-superoxide intermediate with C2 of the indole of Trp, followed by (2) ring-closure via homolytic O-O cleavage to give epoxide and ferryl-oxo (Cpd II) intermediates, (3) acid-catalyzed regiospecific ring-opening of the epoxide, (4) oxo-attack, and (5) finally C-C bond cleavage concerted with back proton transfer. The involvement of dual oxidants, ferric-superoxide and ferryl-oxo (Cpd II) intermediates, is proposed to be responsible for the dioxygenase reactivity in bacterial TDO. In particular, the not-well-recognized ferric-superoxide porphyrin intermediate is found to be capable of reacting with pi-systems via direct radical addition, an uncommon dioxygen activation in the hemes. The comparison between Xanthomonas campestris TDO and some heme as well non-heme oxygenases is also discussed.

Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation

Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular β-helix with six loops of 18 or more residues that lies alongside an α-helix. β-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the β-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of β-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.

Crystal Structure of 1-Aminocyclopropane-1-carboxylate Deaminase from Hansenula saturnus

The pyridoxal 5′-phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) catalyzes a reaction that involves a ring opening of cyclopropanoid amino acid, yielding α-ketobutyrate and ammonia. Unlike other PLP-dependent enzymes, this enzyme has no α-hydrogen atom in the substrate. Thus, a unique mechanism for the bond cleavage is expected. The crystal structure of ACCD fromHansenula saturnus has been determined at 2.0 Å resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers. The model was built on the electron density map, which was obtained by the density averaging of multiple crystal forms. The final model was refined to an R-factor of 22.5% and an R free-factor of 26.8%. The ACCD folds into two domains, each of which has an open twisted α/β structure similar to the β-subunit of tryptophan synthase. However, in ACCD, unlike in other members of the β family of PLP-dependent enzymes, PLP is buried deep in the molecule. The structure provides the first view of the catalytic center of the cyclopropane ring opening.