Sterols and other triterpenoids: source specificity and evolution of biosynthetic pathways

Squalene–hopene cyclase: catalytic mechanism and substrate recognition

Squalene-hopene cyclase: catalytic mechanism and substrate recognition

Terpenoids comprise the largest class of natural products, with nearly 50000 known members. Underlying the astounding structural variation within this class of compounds are the diverse carbon backbone structures formed by terpene synthACHTUNGTRENNUNGases/cyclases. These enzymes mediate complex electrophilic cy ACHTUNGTRENNUNGclizations and/or rearrangements that create these diverse skeletal structures from relatively simple acyclic isoprenoid precursors. However, while the cyclization of triterpenes and many diterpenes are mechanistically similar in that they are initiated by protonation of a C=C double bond, identification and analysis of the genes for the relevant enzymes has revealed that there is no corresponding phylogenetic relationship. In particular, rather than being related to triterpene cyclases, these diterpene cyclases were found to be homologous to “lower” terpene synthases (TPS), which typically initiate catalysis by ionization of the allylic diphosphate ester bond in acyclic isoprenoid substrates, such as the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP, 1). Accordingly, TPSs can be divided into two mechanistically distinct groups, the prevalent class I enzymes, which catalyze diphosphate ionization-initiated reactions, and atypical class II enzymes, which catalyze protonation-initiated cyclization reactions. 3] Structural and mechanistic studies of the common class I TPS enzymes have demonstrated that these synthases contain a characteristic aspartate-rich DDXXD motif that binds divalent metal ions required for catalysis of diphosphate ionization. The first class II TPS identified, ent-copalyl/labdadienyl diphosphate (ent-CPP, 2) synthase (CPS) from Arabidopsis thaliana (AtCPS), was found to lack this DDXXD motif, and instead contained a separately placed DXDD motif. Despite the lack of any other homology, it was suggested that the AtCPS DXDD motif is important for class II catalysis based on the occurrence of a similar motif in previously identified squalene–hopene (triterpene) cyclases (SHC). Structural and mechanistic studies have demonstrated that the DXDD motif in SHC, and the corresponding VXDC motif in oxidosqualene (sterol) cyclases (OSC), initiate cyclization with the “middle” aspartate, which is presumed to act as the catalytic acid. The other conserved residues are thought to “activate” this aspartate for protonation of the terminal C=C double bond of squalene (SHC) or corresponding epoxide ring of oxidosqualene (SHC or OSC). Class II TPS also characteristically contain a DXDD motif, and mutational analysis has demonstrated that the DXDD motif in the bifunctional (i.e. , class I and II) TPS abietadiene synthase (AS) is required for class II activity, which is consistent with the suggestion that the DXDD motif in class II TPS also plays a role in initiating cyclization by protonating the terminal C=C double bond of GGPP. However, these studies also reported two important differences between the class II activity of AS and SHC/OSC. First, the class II activity of AS requires divalent metal ions (preferably Mg), whereas SHC and OSC do not. Second, aza analogues that mimic the initial carbocations formed by protonation are very effective inhibitors of the class II activity of AS, but not SHC/OSC. Specifically, 14,15-dihydro-15-azaGGPP (15-azaGGPP, 3) exhibits approximately nanomolar affinity for the class II active site of AS, but the equivalent 2,3-dihydro-2-azasqualene exhibits much weaker (approximately micromolar) affinity for SHC/OSC. These mechanistic differences, coupled with the lack of homology outside of this short motif, leave the role of the class II TPS DXDD motif in question. In particular, the DXDD motif represents a potential divalent metal binding site and synergistic Mg–GGPP substrate inhibition effects have been interpreted to suggest a role for these aspartates in Mg binding, as well as alternative GGPP binding modes. Despite the importance of these enzymes in catalyzing the committed step in the biosynthesis of the large family of labdane-related diterpenoid natural products (which contains more than 5000 known members), no direct evidence for the exact role of the DXDD motif in class II TPS catalysis has yet been presented. To address this fundamental question, we report here mechanistic studies utilizing the known epoxy analogue of 1, ( )14,15-oxidogeranylgeranyl diphosphate (oxidoGGPP, 4) wherein the C14,15 double bond normally protonated to initiate cyclization is replaced by an epoxide ring (Scheme 1), in combination with site-directed mutational analysis of a recombinant pseudomature version of AtCPS (rAtCPS). It seems reasonable to presume that oxidoGGPP will be more susceptible to protonation than GGPP given the known greater reactivity of oxidosqualene relative to squalene. The increased basicity and strain energy of the epoxide relative to the C=C double bond translates into a less stringent requirement for activation of the catalytic acid. Thus, SHC, with its DXDD motif, can cyclize (i.e. , protonate) either oxidosqualene or squalene, while OSC, with the less acidic VXDC motif, can cyclize/protonate only oxidosqualene and not squalene. Structural analysis of both SHC and OSC has indicated that the absolutely conserved “middle” aspartate acts as the catalytic acid, 14] a conclusion confirmed by various mechanistic and mutational studies. Of particular interest, mutation of the “last” aspartate of the DXDD motif in SHC led to compromised enzymes that were able to readily cyclize oxidosqualene, which has a more easily protonated epoxide ring, but were severely impaired with squalene, which contains the less basic [a] S. Prisic, Prof. Dr. R. J. Peters Department of Biochemistry, Biophysics, and Molecular Biology Iowa State University, Molecular Biology Building Ames, IA 50011 (USA) Fax: (+1)515-294-0453 E-mail : rjpeters@iastate.edu [b] Dr. J. Xu , Prof. Dr. R. M. Coates Department of Chemistry, University of Illinois 600 South Mathews Avenue, Urbana, IL 61801 (USA) [c] Dr. J. Xu Current address: Neurogen Corp. Branford, CT 06405 (USA)

Probing the Role of the DXDD Motif in Class II Diterpene Cyclases

Inulin-based polymer coated SPIONs as potential drug delivery systems for targeted cancer therapy

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

Karl Poralla

Papers

Rationally designed inhibitors as tools for comparing the mechanism of squalene-hopene cyclase with oxidosqualene cyclase.