Engineering the Prenyltransferase Domain of a Bifunctional Assembly-Line Terpene Synthase

Abstract

Copalyl diphosphate (CPP) synthase from Penicillium verruculosum (PvCPS) is a bifunctional diterpene synthase with both prenyltransferase and class II cyclase activities. The prenyltransferase α 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 βγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αβγ)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 α 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 Å, 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.

The biosynthesis of the odorant 2-methylisoborneol is compartmentalized inside a protein shell

“…As we now recognize Sg Enc to be a 2-MIBS-loaded encapsulin nanocompartment, we suspected that if Sg Enc is able to bind cAMP via its CBDs, this may result in a conformational change of the two-fold pores which in turn could control 2-MIB production by potentially restricting substrate flux across the encapsulin shell. To test this hypothesis, we monitored the activity of encapsulated and free 2-MIBS through a coupled assay with the GPP-methylating Sg MT in the presence or absence of cAMP [66][67][68] . We observed sigmoidal saturation curves (Supplementary Fig.…”

Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis

“…[16][17][18][19][20] These catalytic domains are connected by flexible polypeptide linkers that hinder crystallization, but individual domains can be crystallized for structure determination. [21][22][23] For the visualization of full-length enzymes in solution, small-angle X-ray scattering yields low-resolution molecular envelopes showing general domain architecture and quaternary structure. 21,22 More recently, cryo-electron microscopy (cryo-EM) reveals higherresolution images of oligomers comprised of unusual combinations of ordered and disordered catalytic domains.…”

Computer-Informed Engineering: A New Class I Sesquiterpene Synthase JeSTS4 for the Synthesis of an Unusual C10-(S)-Bicyclogermacrene

“…However, the catalytic efficiency of TPSs is generally low, which becomes the bottleneck for using them as biocatalysts for large-scale production of terpenoids in the industry. Efforts towards optimizing the enzyme activities of TPSs to enhance the yields of diverse products such as mono-, sesqui-, or diterpenoids have been reported. − …”