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

Abstract

Staurosporine isolated fromThe 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.heme ͉ staurosporine ͉ rebeccamycin ͉ secondary metabolism S taurosporine and rebeccamycin (Fig. 1A) are natural products that have attracted much attention because of their strong inhibitory activity for protein kinase or DNA topoisomerase, which makes them therapeutically important anticancer agents. These natural products are members of a family of indolocarbazole alkaloids which have a similar structure, including an indole[2,3-a]carbazole core with a C-N linkage to a sugar moiety.The staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and the rebeccamycin biosynthetic gene cluster from Lechevalieria aerocolonigenes (39243; American Type Culture Collection, Manassas, VA) have been cloned and characterized. The staurosporine biosynthetic gene cluster consists of 15 ORFs spanning 22 kb (1), and the rebeccamycin biosynthetic gene cluster consists of 11 genes spanning 17 kb (2, 3). Gene disruption and/or heterologous gene expression experiments revealed that four genes (staO, staD, staP, and staC in Streptomyces sp. TP-A0274 and the homologous genes rebO, rebD, rebP, and rebC in L. aerocolonigenes) are responsible for the biosynthesis of the indolocarbazole skeleton (2, 3). In staurosporine biosynthesis, StaO initiates the synthesis by catalyzing the reaction of tryptophan to the imine form of indole-3-pyruvic acid (IPA imine), and StaD then catalyzes the coupling of two molecules of IPA imine to yield chromopyrrolic acid (CPA). Finally, the key skeleton structure referred to as the indolocarbazole core is constructed through the following two oxidation steps by StaP and StaC (2, 4) (Fig. 1B).StaP (CYP245A1) is a member of the cytochrome P450 family (5), which includes heme enzymes involved in steroid hormone biosynthesis, drug metabolism, and many other physiologically

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

“…In CYP StaP, the helical loop is expanded into two larger loops that protrude from the enzyme surface. One of these, from Arg-67 to Val-81, was proposed to be part of a flexible substrate entry site for the substrate chromopyrrolic acid, a molecule of which was bound at this region (29). The marked divergence of structure may account for the output derived from the molecular replacement solution (see "Experimental Procedures"), in which the protein chains constituting the proximal heme portion of the protein, which overlap very well, were described in the solution, yet the distal secondary elements were not, and hence required tracing using BUCCANEER (20).…”

“…4). Significantly, Met-394, the tip of which is 8.7 Å from the heme iron, is one of a pair of amino acid residues, the other of which is Ala-395, which directly replaces the pair, usually an acidic residue followed by threonine (or serine), illustrated by Glu-257/ Thr-258 in CYP StaP (29), which is well conserved among CYPs known to catalyze oxygenation reactions, in which they are thought to play a critical role in oxygen activation yet absent, for example, in the nitric-oxide reductase P450nor (32). Met-394 is also isolated on the Ramachandran plot for XplAheme with values for ⌽ and ⌿ angles of Ϫ84.9°and Ϫ74.3°, respectively, on the verge of the allowed region.…”

Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis

“…A biradical mechanism has been suggested for other P450-catalyzed C-C coupling reactions: it involves the highly electrophilic compound I (CpdI; Fe IV A Oϩ⅐) as depicted in Fig. 5 (28,29). CpdI can promote the formation of two tyrosyl radicals, as demonstrated with the horseradish peroxidase, another hemecontaining protein forming CpdI as the reactive species (30,31).…”

Crystal Structure of VioE, a Key Player in the Construction of the Molecular Skeleton of Violacein

“…Compound X has been predicted to have two carboxyl groups and two indole rings (8), because both the precursor of the substrate and CPA have two carboxyl groups and two indole rings. Thus, electrostatic and hydrophobic interactions are expected to play a role in the binding between compound X and VioE as has been observed between CPA and StaP, an enzyme in the staurosporine biosynthetic pathway that converts CPA into an indolocarbazole core (27). These observations suggest that the active site of this enzyme should contain a positively charged residue(s).…”