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

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 ßγ domain interface to generate CPP. The prenyltransferase α domain mediates oligomerization to form a 648-kD (αßγ)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.

Rules for the design of aza-glycine stabilized triple-helical collagen peptides.

The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen's repeating (Glycine-Xaa-Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position. For the first time, we present a set of design rules for azGly-stabilized triple-helical collagen peptides, accounting for all canonical amino acids in the Xaa and Yaa positions adjacent to an azGly residue, and extend these rules using multiple azGly residues. To gain atomic level insight into these new rules we present two high-resolution crystal structures of collagen triple helices, with the first peptoid-containing collagen peptide structure. In conjunction with biophysical and computational data, we highlight the critical importance of preserving the triple helix geometry and protecting the hydrogen bonding network proximal to the azGly residue from solvent. Our results provide a set of design guidelines for azGly-stabilized triple-helical collagen peptides and fundamental insight into collagen structure and stability.