7s0m
From Proteopedia
Crystal structure of Penicillium verruculosum copalyl diphosphate synthase (PvCPS) alpha prenyltransferase domain variant, S723T, bound with non-productive isopentenyl diphosphate
Structural highlights
FunctionPVCPS_TALVE Bifunctional terpene synthase that possesses both prenyltransferase and type II terpene cyclase activity, converting isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into geranylgeranyl diphosphate (GGPP) and further converting GGPP into copalyl diphosphate, respectively.[1] [2] Publication Abstract from PubMedCopalyl diphosphate (CPP) synthase from Penicillium verruculosum (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase alpha domain catalyzes the condensation of C5 dimethylallyl diphosphate with three successively added C5 isopentenyl diphosphates (IPPs) to form C20 geranylgeranyl diphosphate (GGPP), which then undergoes a class II cyclization reaction at the betagamma domain interface to generate CPP. The prenyltransferase alpha domain mediates oligomerization to form a 648-kD (alphabetagamma)6 hexamer. In the current study, we explore prenyltransferase structure-function relationships in this oligomeric assembly-line platform with the goal of generating alternative linear isoprenoid products. Specifically, we report steady-state enzyme kinetics, product analysis, and crystal structures of various site-specific variants of the prenyltransferase alpha domain. Crystal structures of the H786A, F760A, S723Y, S723F, and S723T variants have been determined at resolutions of 2.80, 3.10, 3.15, 2.65, and 2.00 A, respectively. The substitution of S723 with bulky aromatic amino acids in the S723Y and S723F variants constricts the active site, thereby directing the formation of the shorter C15 isoprenoid, farnesyl diphosphate. While the S723T substitution only subtly alters enzyme kinetics and does not compromise GGPP biosynthesis, the crystal structure of this variant reveals a nonproductive binding mode for IPP that likely accounts for substrate inhibition at high concentrations. Finally, mutagenesis of the catalytic general acid in the class II cyclase domain, D313A, significantly compromises prenyltransferase activity. This result suggests molecular communication between the prenyltransferase and cyclase domains despite their distant connection by a flexible polypeptide linker. Engineering the Prenyltransferase Domain of a Bifunctional Assembly-Line Terpene Synthase.,Ronnebaum TA, Eaton SA, Brackhahn EAE, Christianson DW Biochemistry. 2021 Oct 5. doi: 10.1021/acs.biochem.1c00600. PMID:34609847[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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