Crystal Structures and Catalytic Mechanism of Cytochrome P450 Stap That Produces the Indolocarbazole Skeleton

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

Masatomo Makino*†, Hiroshi Sugimoto*, Yoshitsugu Shiro*, Shumpei Asamizu‡, Hiroyasu Onaka‡§¶, and Shingo Nagano*¶

*Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Hyogo 679-5148, Japan; †Department of Life Science, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297, Japan; and ‡Department of Biotechnology, Faculty of Engineering and §Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan

Edited by Thomas L. Poulos, University of California, Irvine, CA, and accepted by the Editorial Board June 4, 2007 (received for review April 3, 2007)

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 ␲–␲ 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 dis- cussed 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.

heme ͉ staurosporine ͉ rebeccamycin ͉ secondary metabolism

taurosporine and rebeccamycin (Fig. 1A) are natural prod- Sucts that have attracted much attention because of their Fig. 1. Chemical structures of indolocarbazole and related compounds. (A) strong inhibitory activity for protein kinase or DNA topoisom- Staurosporine and rebeccamycin. (B) CPA and three aglycon products, K252c, erase, which makes them therapeutically important anticancer 7-hydroxy-K252c, and arcyriaﬂavin A. agents. These natural products are members of a family of indolocarbazole alkaloids which have a similar structure, includ- ing an indole[2,3-a]carbazole core with a C–N linkage to a sugar bazole core is constructed through the following two oxidation moiety. steps by StaP and StaC (2, 4) (Fig. 1B). The staurosporine biosynthetic gene cluster from Streptomyces StaP (CYP245A1) is a member of the cytochrome P450 family BIOCHEMISTRY sp. TP-A0274 and the rebeccamycin biosynthetic gene cluster (5), which includes heme enzymes involved in steroid hormone from Lechevalieria aerocolonigenes (39243; American Type Cul- biosynthesis, drug metabolism, and many other physiologically ture Collection, Manassas, VA) have been cloned and charac- terized. The staurosporine biosynthetic gene cluster consists of 15 ORFs spanning 22 kb (1), and the rebeccamycin biosynthetic Author contributions: H.O. and S.N. designed research; M.M., S.A., and H.O. performed research; M.M., H.S., Y.S., and S.N. analyzed data; and M.M., H.S., H.O., and S.N. wrote the gene cluster consists of 11 genes spanning 17 kb (2, 3). Gene paper. disruption and/or heterologous gene expression experiments The authors declare no conﬂict of interest. revealed that four genes (staO, staD, staP, and staC in Strepto- This article is a PNAS Direct Submission. T.L.P. is a guest editor invited by the Editorial Board. myces sp. TP-A0274 and the homologous genes rebO, rebD, rebP, Abbreviations: P450 or CYP, cytochrome P450; CPA, chromopyrrolic acid. and rebC in L. aerocolonigenes) are responsible for the biosyn- Data deposition: Atomic coordinates and structure factors have been deposited in the thesis of the indolocarbazole skeleton (2, 3). In staurosporine Protein Data Bank, www.pdb.org (PDB ID codes 2Z3T and 2Z3U). biosynthesis, StaO initiates the synthesis by catalyzing the reac- ¶To whom correspondence may be addressed. E-mail: [email protected] or onaka@ tion of tryptophan to the imine form of indole-3-pyruvic acid pu-toyama.ac.jp. (IPA imine), and StaD then catalyzes the coupling of two This article contains supporting information online at www.pnas.org/cgi/content/full/ molecules of IPA imine to yield chromopyrrolic acid (CPA). 0702946104/DC1. Finally, the key skeleton structure referred to as the indolocar- © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702946104 PNAS ͉ July 10, 2007 ͉ vol. 104 ͉ no. 28 ͉ 11591–11596 Downloaded by guest on October 1, 2021 Fig. 2. Crystal structure of substrate-free (A) and -bound (B) form of StaP and overlay of two forms (C). (A and B) Helices and ␤-sheets are shown in ribbon and arrows, respectively. Heme groups, the imidazole molecule in A and the CPA molecules in B are shown in red, cyan, and blue sticks, respectively. The secondary structure elements are labeled according to the nomenclature for the typical P450 fold. (C) Superposition of C␣ traces of substrate-free (light blue) and -bound (green) StaP. The heme groups are shown as sticks. StaP undergoes a signiﬁcant conformational changes at the BЈ1/BЈ2 connecting loop (highlighted in red) and the heme group moves toward helix I upon CPA binding.

important monooxygenation reactions. Escherichia coli recom- pocket near the heme. Similar open/closed conformations were binant StaP converts CPA into three different indolocarbazole also reported for CYP119 (14) and P450BM-3 (15). Substrate compounds: K252c, arcyriaflavin A, and 7-hydroxy-K252c, and binding shifts the heme by 1.3 Å toward helix I [Fig. 3; and see the additional presence of StaC directs formation of a single supporting information (SI) Fig. 6]. Relocation of some of the product, K252c (Fig. 1B) (6). residues in the substrate-binding pocket was also observed. Biochemical characterizations of the biosyntheses of stauro- Upon substrate binding, Ser-186 shifts its position by a distance sporine or rebeccamycin have been reported for several enzymes of 1.9 Å toward the substrate, and the side chains of Arg-67 and (7–10). However, until now, the only crystal structure available Gln-183 also swing toward the substrate-binding pocket, making was that of RebH which provides 7-chlorotryptophan, a precur- interactions with CPA. Hydrophobic residues, Trp-97, Phe-100, sor of rebeccamycin (11). Here, we report 2.4-Å and 1.9-Å and Phe-403, change their positions for optimization of the resolution crystal structures of StaP in the presence and absence substrate interactions. of the substrate CPA, respectively. The structure of the complex with the substrate of StaP provides structural insights into the Substrate Conformation and Binding. Unexpectedly, there are three process of substrate recognition and the molecular mechanism CPA molecules observed in the substrate-bound form (Fig. 2B). of indolocarbazole core formation, which includes the steps of One molecule is located in the substrate-binding pocket, which aryl–aryl coupling and oxidative decarboxylation. is lined by the heme, helices BЈ2 and I, and a loop between F and G helices. Another CPA molecule is found between ␤-3,1 and Results and Discussion helix B of another symmetry-related P450 molecule of the crystal Overall Structures and Structural Comparison. Fig. 2 shows the lattice. The third CPA is located between helix BЈ1 and ␤-1,2. overall crystal structure of substrate-free and -bound forms of Although the functional significance of the latter two molecules StaP determined in the ferric (Fe3ϩ) form at resolutions of 1.9 is unclear, and the binding of these two additional molecules Å and 2.4 Å, respectively. The core portion of the monomer has might represent a crystallographic artifact, the site between helix a typical prism-like P450 fold and is divided into ␣- and ␤-rich BЈ1 and ␤-1 is a plausible entry point for CPA because this region domains. StaP has 12 helices (A–L) that are known components is flexible in the substrate-free form and has been proposed to of P450 structures and two ␤-sheets. A kink is formed in the be a substrate entry site for several P450s (16, 17). middle of the I helix where it contains a highly conserved Thr The simulated annealing omit map (Fig. 3C) obviously shows residue and an adjacent acidic residue [Thr-258 and Glu-257, that CPA adopts a twisted butterfly-shaped conformation in the respectively (Fig. 3)], which play critical roles in the process of active site. The average crystallographic temperature factor of oxygen activation (12, 13). A loop preceding helix L provides the the substrate in the active site is 14 Å2, indicating that the proximal heme ligand Cys-364. substrate has high occupancy and a well ordered conformation. Comparisons of the substrate-free form with the substrate- Our structure of the substrate complex defines the molecular bound form revealed changes in the structure predominately at mechanism of substrate recognition by StaP. The CPA molecule the known highly variable regions and substrate recognition sites in the substrate-binding cavity is nearly perpendicular to the (Fig. 2C). In the substrate-free structure, residues between the heme plane and has interactions with residues in helices F, BЈ2, B and C helices and other loop regions cannot be located in the the ␤-1,4 sheet and the ␤-3,1/␤-3,2 connecting loop (Figs. 2B and 2Fo-Fc electron density map. This is most likely because of 3). The hydrogen-bonding interactions between the two carboxyl disorder. In the substrate-bound form, the residues connecting groups of CPA and surrounding polar groups are particularly the B and C helices are relatively well ordered, with an average important interactions. The carboxyl group that faces the heme temperature factor of 30.1 Å2 (shown as red in Fig. 2C), (proximal carboxyl) is hydrogen bonded to the main-chain amide indicating that substrate binding causes structural changes at nitrogen atom of Thr-305, the guanidium group of Arg-67, and these regions, and, in the substrate-free form, StaP exhibits a the 7-propionate of the heme (Fig. 3A). The other carboxyl more open conformation. Such an open conformation observed group (distal carboxyl group) is hydrogen bonded to hydroxyl in the substrate-free form might leave the active site relatively groups of Thr-187 and Ser-186 and the main chain amide exposed and enable the substrate to access the substrate-binding nitrogen atom of Thr-187. The pyrrole N1 atom of CPA is free

11592 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702946104 Makino et al. Downloaded by guest on October 1, 2021 Fig. 4. Electrostatic potential map near Trp-191 in cytochrome c peroxidase (A) and the substrate indole rings in StaP–CPA complex (B). The electrostatic potential maps were ﬁrst generated with APBS (41) Tools on PyMOL (40). Each electrostatic potential map around Trp-191 and the substrate in CPA–StaP complex is shown with negative charge in red and positive charge in blue.

complex results in heterolytic cleavage of the heme linked O–O bond, leaving behind compound I (Porϩ⅐Fe4ϩϭO: a ferryl-oxo porphyrin cation radical), which has two oxidizing equivalents. The oxygen atom of compound I is transferred to the substrate to complete the monooxygenation reaction. A unique feature of StaP is that the enzyme catalyzes an intramolecular aryl–aryl coupling and oxidative decarboxylation rather than monooxygenation reaction. Howard-Jones and Walsh have recently reported (6) that StaP cannot catalyze an aryl–aryl coupling reaction for arcyriarubin A, which is ‘‘decarboxylated CPA.’’ Based on this observation, it was suggested that the process of C–C bond formation between two indole rings precedes the oxidative decarboxylation process (6). Consistent with this proposal, the C10 atom of the proximal indole ring points toward the heme iron at a distance of 4.7 Å, which is comparable with the substrate–iron distances in Fig. 3. Substrate-binding site [side view (A) and top view (B)] and simulated- P450cam and P450eryF [4.2 Å and 4.7 Å, respectively (19, 20)]. annealed 2Fo–Fc composite omit map (C) for CPA. Stereo views of CPA bound On the other hand, the two carboxyl groups, which are greater in the active site viewed from side (A) and top (B) of the heme plane are shown. than 8 Å away from the heme iron, are directed toward the CPA, the heme group, and residues involved in substrate recognition are enzyme surface. Therefore, the StaP–CPA complex structure rendered as sticks. Carbon atoms of CPA are colored orange, and carbon atoms also suggests that the initial step of the overall reaction of StaP of the heme are colored yellow. Nitrogen, oxygen, and iron atoms are shown in blue, dark red, and bright red, respectively. The carbon atoms of surround- is the aryl-aryl coupling reaction. ing residues are shown in silver. Portions of the I helix are shown as a green The proximal indole in the StaP-CPA complex is 3.5 Å above ribbon, and the water molecule in the active site is shown as a red sphere. the pyrrole D ring of the heme. A similar relative orientation Potential hydrogen bonds are shown as dotted yellow lines. between the indole and the heme was demonstrated for Trp-191 in the active site of cytochrome c peroxidase (CcP) (21–23), which is a heme-containing enzyme that catalyzes H2O2- of hydrogen-bonding interactions. The two indole rings are held dependent oxidation of ferrous cytochrome c (Fig. 4). After in place by T-shaped ␲–␲ interactions (18) with Trp-97 and treatment with peroxide, CcP can store two oxidizing equivalents Phe-403 and hydrophobic interactions with Val-99, Phe-100, and (CcP compound I, SI Fig. 7B). One equivalent is stored as the Val-402 (Fig. 3B and SI Fig. 6). In addition, the nitrogen atom ferryl-oxo state of the iron (Fe4ϩϭO), and the other equivalent of the distal indole has a hydrogen-bonding interaction with exists as a Trp-191 indole cation radical (21). A normal indole BIOCHEMISTRY Gln-183. The proximal indole nitrogen atom has a hydrogen radical would be readily quenched because of its very high redox bond with a water molecule, which also provides another hy- potential (24). However, Trp-191 has a negative electrostatic drogen bond to the side chains of His-250 and Thr-253. In environment formed by surrounding electronegative residues, summary, the two carboxyl groups of CPA together play a critical which, in turn, stabilizes the tryptophan cation radical as evi- role in anchoring the substrate to the enzyme, whereas several denced by spectroscopic experiments and investigations of mu- T-shaped ␲–␲ interactions, hydrophobic interactions, and hy- tants of CcP (22). As shown in Fig. 4B, a strong negative drogen bonds define the orientation and position of two indole electrostatic potential near the substrate is provided by two moieties of CPA. carboxyl groups of the CPA molecule, two heme propionate groups, and several other electronegative residues. The very Catalytic Mechanism and Relevance to Cytochrome c Peroxidase. In similar structural and electrostatic features of these enzymes the P450 reaction cycle (SI Fig. 7A), the ferric enzyme first binds allow us to propose that compound I in the StaP–CPA complex the substrate and initiates a subsequent one-electron reduction first removes one electron from the proximal indole ring to by a redox-partner to generate the ferrous (Fe2ϩ) reduced form. generate an indole cation radical and ferryl-oxo heme Molecular oxygen binding to the heme iron of the reduced (Fe4ϩϭO), which is equivalent to CcP compound I (step 1 in Fig. enzyme provides the ferrous P450–oxygen complex. Injection of 5). The CPA molecule is located at a distance 4.7 Å from the one electron and two protons to the active site in the oxygen iron, which is apparently close enough to promote the monoox-

Makino et al. PNAS ͉ July 10, 2007 ͉ vol. 104 ͉ no. 28 ͉ 11593 Downloaded by guest on October 1, 2021 After the occurrence of C–C bond formation by single StaP turnover, decarboxylation and oxygen addition are required to complete the indolocarbazole core formation. In the second turnover, the enzyme might catalyze one-electron oxidation of the aryl–aryl coupled product (the final product in Fig. 5). Subsequent deprotonation of the carboxyl group can lead to decarboxylation, leaving behind a neutral radical that then binds molecular oxygen to introduce oxygen atom(s). This is consistent with the finding that the added oxygen atoms in two of the final products, K252c and arcyriaflavin A, originate from molecular oxygen (6). Steric and electrostatic interactions may assist the decarboxylation reaction. The aryl–aryl coupling process trans- forms the conformation of the indole moiety from the twisted butterfly-shape of CPA into the planar indolocarbazole scaffold. The conformational change would induce decarboxylation to avoid clashes between the carboxyl group and the planar indolocarbazole core. In addition, electronic repulsion between the proximal carboxylate and the 7-propionate of the heme might assist in the decarboxylation process, as was observed for oro- tidine monophosphate decarboxylase (28, 29). The reconstituted StaP turnover system, which includes NADPH, ferredoxin, flavodoxin NADH reductase, and StaP, produces the three indolocarbazole products, K252c, arcyriaflavin A, and 7-hydroxy-K252c. It is particularly interesting to note that the additional presence of StaC gives a single product, K252c (6). Before the in vitro experiments on StaP and StaC, a product-funneling process was proposed for rebeccamycin biosynthesis enzymes, in parallel with the staurosporine biosynthesis system. Disruption of the rebC gene, whose enzyme is involved in the last step of formation of the indolocarbazole Fig. 5. Proposed mechanism for aryl-aryl coupling of CPA by StaP. skeleton in rebeccamycin biosynthesis, revealed that RebP could construct three types of indolocarbazole. This suggests that RebP catalyzes the C–C bond formation and, presumably, ygenation reaction. However, no monooxygenated products oxidative decarboxylation and that RebC directs the formation were observed under in vitro turnover conditions (6). Extensive of a single product (2). hydrogen bonding and aromatic interactions tightly bind the Although StaC has a conserved FAD-binding motif in the substrate to limit the substrate flexibility required for interac- deduced amino acid sequence, StaC, which does not have FAD, tions with compound I. Therefore, StaP favors one-electron can also direct the product flux. This implies that the flavin oxidation rather than hydroxylation of the proximal indole. In cofactor is not necessarily required for this effect and that the the next step, the indole cation radical of CPA then releases a redox reaction of StaC is not essential. This is reminiscent of the proton to form a neutral radical because of the acidity of the effector role of cytochrome b5 on P450, CYP17A, for example (30). Cytochrome b appears to function in directing the route of indole cation radical (pKa ϭ 4.3 (25), step 2). The remaining 5 ferryl-oxo heme of StaP again removes one electron from the steroid biosynthesis between androgens and corticosteroids. substrate, at which point, the proximal indole cation radical is Both cytochromes b5 with and without heme have effector deprotonated (step 3). Intramolecular radical coupling then activity on pregnenolone 17,20-lyase activity (31). Biochemical forms the C–C bond between the two indole rings (step 4). In the and spectroscopic studies on StaC are currently in progress to final stage of the reaction, tautomerization of the six-ring species elucidate the function(s) of StaC. provides the aromatized six-ring system of the indolocarbazole Implications for Structures and Catalysis by RebP and AtmP. Our core (step 5). Alternatively, an indole-neutral radical could structure of the substrate complex of StaP also has significant directly create the C–C bond, leaving a neutral radical on the implications for structure and substrate recognition by homol- proximal indole ring (step 3Ј). An additional one-electron ogous P450s, RebP (2, 3), and AtmP (32). RebP and AtmP, oxidation and deprotonation of the proximal indole ring would Ͼ Ј whose amino acid sequences share 50% identity with StaP (SI then produce the indolocarbazole core (step 4 ). In either Fig. 8), catalyze the aryl–aryl coupling reaction and oxidative reaction pathway, the indole cation radical intermediate is a key decarboxylations of 11,11Ј-dichlorochromopyrrolic acid (CCA) catalytic intermediate, and ferric enzyme is regenerated after and 11-chlorochromopyrrolic acid (Cl-CPA), respectively. A completion of the coupling process. comparison of the amino acid sequences of these two homolo- An aryl–aryl coupling reaction is also reported for P450 158A2 gous P450 enzymes reveals that almost all key active site residues that produces flaviolin dimer (26) and P450 OxyC that is for substrate recognition and catalysis are conserved. Taken involved in vancomycin biosynthesis (27). Based on the crystal together, CCA in RebP and Cl-CPA in AtmP have the same structure of CYP158A2 complexed with the substrates, Zhao et binding mode found in the CPA-StaP complex, and the reaction al. (26) proposed that the aryl–aryl coupling proceeds by means should proceed through the same mechanism proposed for StaP. of direct hydrogen abstraction or direct bond formation with It is, thus, not surprising that StaP can produce indolocarbazole compound I. Although these mechanisms can explain the reac- product from CCA, the substrate for RebP (6). tion by P450 158A2, they are an unlikely for the StaP reaction Salas and coresearchers reported on the combinatorial bio- because, as mentioned, the extensive substrate-StaP interaction synthesis of indolocarbazole compounds (4). Two products, limits the substrate dynamics required for direct interaction 9-chloro-CPA and 9,9Ј-dichloro-CPA, were obtained by using between the substrate and compound I. rebO, rebD, and pyrH, which codes for tryptophan halogenase.

11594 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702946104 Makino et al. Downloaded by guest on October 1, 2021 Coexpression of rebP and rebC or rebP and staC enables the of 22% PEG3350, 50 mM potassium fluoride, and 100 mM synthesis of 9-chloro-K252c or 9-chloroarcyriaflavin A. On the imidazole (pH 8.0). The crystal used for the diffraction exper- contrary, however, symmetric indolocarbazole products were iment was cryoprotected by the reservoir solution containing not reported to be detected in the same biosynthesis system (SI 20% ethylene glycol. X-ray data were collected by using the Fig. 9A) (4). We presumed that there is bias for these products ADSC Quantum 210 CCD detector in BL44B2 at SPring-8, produced by RebP, which is induced by the substrate-binding Japan. The HKL2000 (34) program was used for integration of pocket. The chlorine atom of the proximal indole would be the diffraction intensity and scaling. The substrate-free crystal is expected to clash with 6-CH of the heme, whereas flexible loop 3 a pseudomerohedral twin crystal of space group P21, a ϭ 89.6, regions around the distal indole ring allow accommodation of b ϭ 78.7, c ϭ 145.6 Å, ␤ ϭ 107.9°. Four StaP monomers exist in this bulky group. Therefore, these P450s can produce only the asymmetric unit. Because the unit cell parameters satisfy the asymmetrically substituted indolocarbazoles. In addition, asym- condition ccos ␤ ϭϪa/2 and because the four molecules are metric indolocarbazole skeletons are often observed in natural quite symmetric, the crystal can emulate a pseudo-P2 2 2 cells compounds; Indocarbazostatin and indocarbazostatins B, C, and 1 1 1 with a ϭ 44.8, b ϭ 78.7, and c ϭ 138.6 Å with one monomer in D isolated from Streptomyces sp. TA-0403 contain a hydroxyl an asymmetric unit. Two of three pseudo-cell axes coincide with group at C3 position of the indolocarbazole skeleton (SI Fig. 9B). a or b axis of P21. The crystal also exhibits a pseudo-C2221 lattice However, compounds modified at both C3 and C9 positions have ϭ ϭ ϭ not been isolated (33). These two facts, the existence of asym- with cell parameters a 89.6, b 277.1, and c 78.7 Å and suggests some resemblance to the pseudomerohedral twin crys- metric natural indolocarbazoles, and the highly conserved amino ␥␦ acids in the active site among StaP, RebP, and AtmP, imply that tal of T cell ligand T10 (35). The initial phase was calculated the substrate-binding architecture and the substrate-binding from the multiwavelength anomalous diffraction data set of mode have limited the diversity of the indolocarbazole com- 2.4-Å resolution by treating it as P212121 in the program SOLVE. pounds produced by natural bacteria or combinatorial biosyn- The atomic model was built automatically by using the program thesis systems. RESOLVE. The model was manually fitted into the density by using the program COOT (36) and refined by using the program Conclusions CNS using the protocol of a hemihedrally twinned crystal of P21 We have determined the crystal structure of substrate-free and with a fixed twin fraction of 0.45 and twin operator of (Ϫh, Ϫk, -bound forms of StaP that catalyzes staurosporine aglycon hϩl). The test set (5% of observed reflections) was selected for formation from CPA. These are the first crystal structures of the cross-validation (37) so that pairs of twin-related reflections enzymes involved in indolocarbazole core formation. StaP at- are in the same set (either working or test) and retained during tains a more ordered conformation upon binding of the sub- all of the refinement steps. The final structure has an Rcryst of strate. Substantial conformational changes are observed at and 22.9% and an Rfree of 27.2% for the detwinned data. Details of around the substrate-binding pocket to optimize interactions the MAD data analyses and model refinements are described in with the enzyme. The StaP–CPA complex structure revealed SI Materials and Methods. that CPA has a twisted-butterfly shape in the active site, which The crystal of the CPA-bound form was obtained under the is perpendicular to the heme plane. Interestingly, it is postulated conditions of 2% PEG4000, 50 mM Bis-Tris (pH 6.8), 100 mM that the enzyme involved in biosynthesis of the anticancer agent MgCl2, 10 mM MnCl2, and 5 mM CPA. The crystal belongs to catalyzes the reaction by means of an indole cation radical the orthorhombic space group P212121, with cell dimensions of intermediate, which is similar to an intermediate of CcP that a ϭ 44.6, b ϭ 66.6, and c ϭ 136.8 Å, containing one monomer oxidizes an electron transfer protein. The structures we solved per asymmetric unit. The phase was obtained with the molecular have also provided valuable information necessary for elucida- replacement method by using the coordinates of the substrate- tion of the mechanism of indolocarbazole core formation mech- free structure as a search model. After several cycles of refine- anism and for understanding the diverse chemistry performed by ment by using CNS and manual refitting, CPA was fitted into the P450s. The present results will open the door for design of indolocarbazole biosynthesis systems that should produce a wide Fo-Fc map. The parameter and topology files for CPA were variety of indolocarbazole compounds. generated by using the PRODRG server (38). Further refinement enabled us to build residues 69–87, 191–193, and 215–223, Materials and Methods which are invisible in the substrate-free form because of disor- Protein Expression and Purification. The coding region for StaP der. All crystallographic statistics are shown in SI Table 1. from Streptomyces sp. TP-A0274 was cloned into expression Figures were created with MOLSCRIPT (39) and PYMOL (40). vector pET26b(ϩ) (Novagen, San Diego, CA), with a C-terminal ϫ E. coli We thank Dr. Seiji Yamada and Mr. Taiji Matsu for their assistance in

6 histidine tag, and overexpressed in strain BL21(DE3). BIOCHEMISTRY The protein was purified by using a nickel-affinity column and data collection at SPring-8 and Profs. Yoshihito Watanabe (Nagoya anion-exchange chromatography. The purified StaP gave a University, Nagoya, Japan) and Hiroshi Fujii (Institute for Molecular single band on SDS/PAGE. Before crystallization, the sample Science, Okazaki, Japan) for their suggestions on the catalytic mechanism. This work was supported, in part, by the Structural Biology buffer was exchanged into 25 mM sodium phosphate (pH 7.4) Research Program at RIKEN (to Y.S.), the Molecular Ensemble Re- and 25 mM NaCl, concentrated to 30–40 mg/ml. More detailed search Program at RIKEN (to Y.S.), a grant-in-aid for Scientific information on the preparation of CPA is described in SI Research on Priority Area from the Ministry of Education, Science, Materials and Methods. Culture, and Sports of Japan (to S.N., Y.S., and H.O.), and an Industrial Technology Research grant in 2006 from the New Energy and Industrial Crystallization, Data Collection, and Structure Determination. The Technology Development Organization (NEDO) of Japan (to H.O.). substrate-free form of the enzyme was crystallized by using the M.M. is supported by the Japan Society for the Promotion of Science hanging-drop vapor-diffusion method with a reservoir solution Research Fellowship for Young Scientists.

1. Onaka H, Taniguchi S, Igarashi Y, Furumai T (2002) J Antibiot 55:1063–1071. 4. Sanchez C, Zhu LL, Brana AF, Salas AP, Rohr J, Mendez C, Salas JA (2005) 2. Onaka H, Taniguchi S, Igarashi Y, Furumai T (2003) Biosci Biotechnol Biochem Proc Natl Acad Sci USA 102:461–466. 67:127–138. 5. Ortiz de Montellano PR, ed (2005) Cytochrome P450: Structure, Mechanism, 3. Sanchez C, Butovich IA, Brana AF, Rohr J, Mendez C, Salas JA (2002) Chem and Biochemistry (Kluwer/Plenum, New York). Biol 9:519–531. 6. Howard-Jones AR, Walsh CT (2006) J Am Chem Soc 128:12289–12298.

Makino et al. PNAS ͉ July 10, 2007 ͉ vol. 104 ͉ no. 28 ͉ 11595 Downloaded by guest on October 1, 2021 7. Howard-Jones AR, Walsh CT (2005) Biochemistry 44:15652–15663. 24. DeFelippis MR, Murthy CP, Faraggi M, Klapper MH (1989) Biochemistry 8. Nishizawa T, Gruschow S, Jayamaha DHE, Nishizawa-Harada C, Sherman DH 28:4847–4853. (2006) J Am Chem Soc 128:724–725. 25. Posener ML, Adams GE, Wardman P, Cundall RB (1976) J Chem Soc Faraday 9. Nishizawa T, Aldrich CC, Sherman DH (2005) J Bacteriol 187:2084–2092. Trans I 72:2231–2239. 10. Asamizu S, Kato Y, Igarashi Y, Furumai T, Onaka H (2006) Tetrahedron Lett 26. Zhao B, Guengerich FP, Bellamine A, Lamb DC, Izumikawa M, Lei L, Podust 47:473–475. LM, Sundaramoorthy M, Kalaitzis JA, Reddy LM, et al. (2005) J Biol Chem 11. Yeh E, Blasiak LC, Koglin A, Drennan CL, Walsh CT (2007) Biochemistry 280:11599–11607. 46:1284–1292. 27. Pylypenko O, Vitali F, Zerbe K, Robinson JA, Schlichting I (2003) J Biol Chem 12. Imai M, Shimada H, Watanabe Y, Matsushima-Hibiya Y, Makino R, Koga H, 278:46727–46733. Horiuchi T, Ishimura Y (1989) Proc Natl Acad Sci USA 86:7823–7827. 28. Appleby TC, Kinsland C, Begley TP, Ealick SE (2000) Proc Natl Acad Sci USA 13. Gerber NC, Sligar SG (1994) J Biol Chem 269:4260–4266. 97:2005–2010. 14. Yano JK, Koo LS, Schuller DJ, Li H, Ortiz de Montellano PR, Poulos TL 29. Wu N, Mo YR, Gao JL, Pai EF (2000) Proc Natl Acad Sci USA 97:2017–2022. (2000) J Biol Chem 275:31086–31092. 30. Schenkman JB, Jansson I (2003) Pharmacol Ther 97:139–152. 15. Li HY, Poulos TL (1997) Nat Struct Biol 4:140–146. 31. Auchus RJ, Lee TC, Miller WL (1998) J Biol Chem 273:3158–3165. 16. Dunn AR, Dmochowski IJ, Bilwes AM, Gray HB, Crane BR (2001) Proc Natl 32. Gao QJ, Zhang CS, Blanchard S, Thorson JS (2006) Chem Biol 13:733–743. Acad Sci USA 98:12420–12425. 33. Feng YF, Mitsuhashi S, Kishimoto T, Ubukata M (2005) J Antibiot 58:759–765. 17. Winn PJ, Ludemann SK, Gauges R, Lounnas V, Wade RC (2002) Proc Natl 34. Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326. Acad Sci USA 99:5361–5366. 35. Rudolph MG, Wingren C, Crowley MP, Chien YH, Wilson IA (2004) Acta 18. Hunter CA, Sanders JKM (1990) J Am Chem Soc 112:5525–5534. Crystallogr D 60:656–664. 19. Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J (1985) J Biol Chem 36. Emsley P, Cowtan K (2004) Acta Crystallogr D Biol Crystallogr 60:2126–2132. 260:16122–16130. 37. Brunger AT (1992) Nature 355:472–475. 20. Cupp-Vickery JR, Poulos TL (1995) Nat Struct Biol 2:144–153. 38. Schuttelkopf AW, van Aalten DM (2004) Acta Crystallogr D 60:1355–1363. 21. Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245: 39. Kraulis PJ (1991) J Appl Crystallogr 24:946–950. 738–740. 40. DeLano WL (2002) The PyMol Molecular Graphics System (DeLano Scien- 22. Fishel LA, Farnum MF, Mauro JM, Miller MA, Kraut J, Liu YJ, Tan XL, tific, San Carlos, CA). Scholes CP (1991) Biochemistry 30:1986–1996. 41. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Proc Natl Acad 23. Erman JE, Yonetani T (1975) Biochim Biophys Acta 393:350–357. Sci USA 98:10037–10041.

11596 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0702946104 Makino et al. Downloaded by guest on October 1, 2021