Use of a head-to-tail peptide cyclase to prepare hybrid RiPPs.

Cyclotides and lanthipeptides are cyclic peptide natural products with promising bioengineering potential. No peptides have been isolated that contain both structural motifs defining these two families, an N-to-C cyclised backbone and lanthionine linkages. We combined their biosynthetic machineries to produce hybrid structures that possess improved activity or stability, demonstrate how the AEP-1 plant cyclase can be utilised to complete the maturation of the sactipeptide subtilosin A, and present head-to-tail cyclisation of the glycocin sublancin. These studies show the plasticity of AEP-1 and its utilisation alongside other post-translational modifications.

Molecular Basis of Iterative C─H Oxidation by TamI, a Multifunctional P450 monooxygenase from the Tirandamycin Biosynthetic Pathway.

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C─H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C─H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C─H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.

Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein.

Despite the growing application of gas-phase measurements in structural biology and drug discovery, the factors that govern protein stabilities and structures in a solvent-free environment are still poorly understood. Here, we examine the solvent-free unfolding pathway for a group of homologous serum albumins. Utilizing a combination of chemical probes and noncovalent reconstructions, we draw new specific conclusions regarding the unfolding of albumins in the gas phase, as well as more general inferences regarding the sensitivity of collision induced unfolding to changes in protein primary and tertiary structure. Our findings suggest that the general unfolding pathway of low charge state albumin ions is largely unaffected by changes in primary structure; however, the stabilities of intermediates along these pathways vary widely as sequences diverge. Additionally, we find that human albumin follows a domain associated unfolding pathway, and we are able to assign each unfolded form observed in our gas-phase data set to the disruption of specific domains within the protein. The totality of our data informs the first detailed mechanism for multidomain protein unfolding in the gas phase, and highlights key similarities and differences from the known solution-phase pathway.