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.
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 His250 residue and the crystal waters, Wat644 and Wat789, 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 Wat644 and Wat789 move about and mediate these events. Experiments with the dichlorinated substrate, CCA, which expels Wat644, show that the enzyme loses its activity. H250A and H250F mutations of P450 StaP show that His250 is important, but in its absence Wat644 and Wat789 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).
In the general stress response of Bacillus subtilis, which is governed by the sigma factor σB, stress signalling is relayed by a cascade of Rsb proteins that regulate σB 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 Mn2+ 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 Mg2+ 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.
Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O2 reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and ...neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4Å resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three α-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.
Gas derivatization of protein crystals is useful not only to analyse gas‐binding proteins but also to solve the phase problem of X‐ray crystallography by using noble gases. However, the gas ...pressurization tools for these experiments are often elaborate and need to release the gas before flash‐cooling. To simplify this step, a procedure using a fine‐needle capillary to mount and flash‐cool protein crystals under the pressurization of gases has been developed. After the crystals are picked up with the capillary, the capillary is sealed with an adhesive and then connected directly to a gas regulator. The quality of the diffraction data using this method is comparable with that of data from conventional pressurization procedures. The preparation of xenon‐derivatives of hen egg‐white lysozyme using this method was a success. In the derivatives, two new xenon binding sites were found and one of their sites vanished by releasing the gas. This observation shows the availability of flash‐cooling under gas pressurization. This procedure is simple and useful for preparing gas‐derivative crystals.
Glycine amide (GlyAd), a typically amidated amino acid, is a versatile additive that suppresses protein aggregation during refolding, heat treatment, and crystallization. In spite of its ...effectiveness, the exact mechanism by which GlyAd suppresses protein aggregation remains to be elucidated. Here, we show the crystal structure of the GlyAd–lysozyme complex by high resolution X-ray crystallographic analysis at a 1.05
Å resolution. GlyAd bound to the lysozyme surface near aromatic residues and decreased the amount of bound waters and increased the mobility of protein. Arg and GlyAd molecules are different in binding sites and patterns from glycerol and related compounds, indicating that decreasing hydrophobic patches might be involved in suppression of protein aggregation.
Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is ...an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe−His stretching (νFe - His) bands at 229 and 221 cm-1, respectively. No time-dependent shift in the νFe - His band of Cgb and Ngb was detected in the 20−1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (νFe - CO, νC - O, δFe - C - O) and (νFe - NO, νN - O, δFe - N - O) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.
Protein microcrystals of less than 10 µm in size are now applicable to X‐ray studies by synchrotron microbeam technology. However, because of their small size, they are difficult to handle and mount. ...In addition, the deterioration of data quality by scattering from the mounting apparatus and crystallization solvent is not negligible. To address these issues, a simple mounting method is proposed using a fine‐needle capillary similar to that used for microinjection in cell biology. In this method, microcrystals are pulled up by capillary action or pipetting, and are held at the tip together with a small amount of cryoprotectant. The quality of the diffraction data using this method is comparable to that of data from conventional cryoloops. This solid apparatus is hopefully suitable for automation of microcrystal handling coupled with optical tweezers.
PDF
