Masatomo Makino

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.

Theoretical and Experimental Studies of the Conversion of Chromopyrrolic Acid to an Antitumor Derivative by Cytochrome P450 StaP: The Catalytic Role of Water Molecules

Chromopyrrolic acid (CPA) oxidation by cytochrome P450 StaP is a key process in the biosynthesis of antitumor drugs (Onaka, H.; Taniguchi, S.; Igarashi, Y.; Furumai, T. Biosci. Biotechnol. Biochem. 2003, 67, 127-138), which proceeds by an unusual C-C bond coupling. Additionally, because CPA is immobilized by a hydrogen-bonding array, it is prohibited from undergoing direct reaction with Compound I, the active species of P450. As such, the mechanism of P450 StaP poses a puzzle. In the present Article, we resolve this puzzle by combination of theory, using QM/MM calculations, and experiment, using crystallography and reactivity studies. Theory shows that the hydrogen-bonding machinery of the pocket deprotonates the carboxylic acid groups of CPA, while the nearby His(250) residue and the crystal waters, Wat(644) and Wat(789), assist the doubly deprotonated CPA to transfer electron density to Compound I; hence, CPA is activated toward proton-coupled electron transfer that sets the entire mechanism in motion. The ensuing mechanism involves a step of C-C bond formation coupled to a second electron transfer, four proton-transfer and tautomerization steps, and four steps where Wat(644) and Wat(789) move about and mediate these events. Experiments with the dichlorinated substrate, CCA, which expels Wat(644), show that the enzyme loses its activity. H250A and H250F mutations of P450 StaP show that His(250) is important, but in its absence Wat(644) and Wat(789) form a hydrogen-bonding diad that mediates the transformation. Thus, the water diad emerges as the minimal requisite element that endows StaP with function. This highlights the role of water molecules as biological catalysts that transform a P450 to a peroxidase-type (Derat, E.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 13940-13949).

High-resolution structure of human cytoglobin: identification of extra N- and C-termini and a new dimerization mode.

Cytoglobin (Cgb) is a recently discovered member of the vertebrate haem-containing globin family. The structure of a new crystal form of wild-type human Cgb (space group C2) was determined at a resolution of 1.68 Angstrom. The results show the presence of an additional helix in the N-terminal residues (4-20) prior to the A helix and an ordered loop structure in the C-terminal region (168-188), while these extended peptides were invisible owing to disorder in the previously reported structures using a P3(2)21 crystal at a resolution of 2.4 Angstrom. A detailed comparison of the two crystal structures shows differences in the conformation of the residues (i.e. Arg84) in the haem environment owing to a different dimeric arrangement.

Crystal structure of the carbon monoxide complex of human cytoglobin

Cytoglobin (Cgb) is a vertebrate heme‐containing globin‐protein expressed in a broad range of mammalian tissues. Unlike myoglobin, Cgb displays a hexa‐coordinated (bis‐hystidyl) heme iron atom, having the heme distal His81(E7) residue as the endogenous sixth ligand. In the present study, we crystallized human Cgb in the presence of a reductant Na2S2O4 under a carbon monoxide (CO) atmosphere, and determined the crystal structure at 2.6 Å resolution. The CO ligand occupies the sixth axial position of the heme ferrous iron. Eventually, the imidazole group of His81(E7) is expelled from the sixth position and swings out of the distal heme pocket. The flipping motion of the His81 imidazole group accompanies structural readjustments of some residues (Gln62, Phe63, Gln72, and Ser75) in both the CD‐corner and D‐helix regions of Cgb. On the other hand, no significant structural changes were observed in other Cgb regions, for example, on the proximal side. These structural alterations that occurred as a result of exogenous ligand (CO) binding are clearly different from those observed in other vertebrate hexa‐coordinated globins (mouse neuroglobin, Drosophila melanogaster hemoglobin) and penta‐coordinated sperm whale myoglobin. The present study provides the structural basis for further discussion of the unique ligand‐binding properties of Cgb. Proteins 2011.

Present status of SPring-8 macromolecular crystallography beamlines

Seven beamlines are operated for macromolecular crystallography (MX) at SPring‐8. The three undulator beamlines are developed for cutting edge target and four bending‐magnet beamlines are developed for high throughput MX. The undulator beamline, BL41XU that provides the most brilliant beam, is dedicated to obtain high quality data even from small‐size and weakly‐diffracting crystals. The minimum beam size at sample position is achieved to 10 μm diameter using a pin‐hole collimator. Its photon flux at wavelength λ = 1.0 A is 2.8×1011 photons/sec. This small beam coupled with irradiation point scanning method is quite useful to take diffraction dataset from small crystals by suppressing the radiation damage. These advanced technologies made a number of difficult protein structure analysis possible, (i.e. Sodium‐potassium ATPase). The bending‐magnet beamlines BL26B1/B2 and BL38B1 provide automatic data collection exploiting the high mobility of the beam. The beamline operation software “BSS,” sample auto‐cha...

Structure of the RsbX phosphatase involved in the general stress response of Bacillus subtilis

In the general stress response of Bacillus subtilis, which is governed by the sigma factor σ(B), stress signalling is relayed by a cascade of Rsb proteins that regulate σ(B) activity. RsbX, a PPM II phosphatase, halts the response by dephosphorylating the stressosome composed of RsbR and RsbS. The crystal structure of RsbX reveals a reorganization of the catalytic centre, with the second Mn(2+) ion uniquely coordinated by Gly47 O from the β4-α1 loop instead of a water molecule as in PPM I phosphatases. An extra helical turn of α1 tilts the loop towards the metal-binding site, and the β2-β3 loop swings outwards to accommodate this tilting. The residues critical for this defining feature of the PPM II phosphatases are highly conserved. Formation of the catalytic centre is metal-specific, as crystallization with Mg(2+) ions resulted in a shift of the β4-α1 loop that led to loss of the second ion. RsbX also lacks the flap subdomain characteristic of PPM I phosphatases. On the basis of a stressosome model, the activity of RsbX towards RsbR-P and RsbS-P may be influenced by the different accessibilities of their phosphorylation sites.