Assessing the Combinatorial Potential of the RiPP Cyanobactin <i>tru</i> Pathway

Ribosomally produced natural products, the RiPPs, exhibit features that are potentially useful in the creation of large chemical libraries using simple mutagenesis. RiPPs are encoded on ribosomal precursor peptides, but they are extensively posttranslationally modified, endowing them with properties that are useful in drug discovery and biotechnology. In order to determine which mutations are acceptable, strategies are required to determine sequence selectivity independently of the context of flanking amino acids. Here, we examined the absolute sequence selectivity of the trunkamide cyanobactin pathway, <i>tru</i>. A series of random double and quadruple simultaneous mutants were synthesized and produced in <i>Escherichia coli</i>. Out of a total of 763 mutated amino acids examined in 325 unique sequences, 323 amino acids were successfully incorporated in 159 sequences, leading to >300 new compounds. Rules for <i>tru</i> sequence selectivity were determined, which will be useful for the design and synthesis of combinatorial biosynthetic libraries. The results are also interpreted in comparison to the known natural products of <i>tru</i> and <i>pat</i> cyanobactin pathways

Crystallographic Evidence of Drastic Conformational Changes in the Active Site of a Flavin-Dependent <i>N</i>‑Hydroxylase

The soil actinomycete <i>Kutzneria</i> sp. 744 produces a class of highly decorated hexadepsipeptides, which represent a new chemical scaffold that has both antimicrobial and antifungal properties. These natural products, known as kutznerides, are created via nonribosomal peptide synthesis using various derivatized amino acids. The piperazic acid moiety contained in the kutzneride scaffold, which is vital for its antibiotic activity, has been shown to derive from the hydroxylated product of l-ornithine, l-<i>N</i>⁵-hydroxyornithine. The production of this hydroxylated species is catalyzed by the action of an FAD- and NAD­(P)­H-dependent <i>N</i>-hydroxylase known as KtzI. We have been able to structurally characterize KtzI in several states along its catalytic trajectory, and by pairing these snapshots with the biochemical and structural data already available for this enzyme class, we propose a structurally based reaction mechanism that includes novel conformational changes of both the protein backbone and the flavin cofactor. Further, we were able to recapitulate these conformational changes in the protein crystal, displaying their chemical competence. Our series of structures, with corroborating biochemical and spectroscopic data collected by us and others, affords mechanistic insight into this relatively new class of flavin-dependent hydroxylases and adds another layer to the complexity of flavoenzymes

Biosynthetic Chlorination of the Piperazate Residue in Kutzneride Biosynthesis by KthP

Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete <i>Kutzneria</i> sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of <i>Kutzneria</i>, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3<i>S</i>)- but not the (3<i>R</i>)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3<i>S</i>,5<i>S</i>)-5-chloropiperazate, distinct from the 3<i>S</i>,5<i>R</i> stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3<i>S</i>,5<i>S</i>)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks