Person:

Nakamura, Hitomi

The termination step is an important source of structural diversity in polyketide biosynthesis. Most type I polyketide synthase (PKS) assembly lines are terminated by a thioesterase (TE) domain located at the Cterminus of the final module, while other PKS assembly lines lack a terminal TE domain and are instead terminated by a separate enzyme in trans. In cylindrocyclophane biosynthesis, the type I modular PKS assembly line is terminated by a freestanding type III PKS (CylI). Unexpectedly, the final module of the type I PKS (CylH) also possesses a C-terminal TE domain. Unlike typical type I PKSs, the CylH TE domain does not influence assembly line termination by CylI in vitro. Instead, this domain phylogenetically resembles a type II TE and possesses activity consistent with an editing function. This finding may shed light on the evolution of unusual PKS termination logic. In addition, the presence of related type II TE domains in many cryptic type I PKS and nonribosomal peptide synthetase (NRPS) assembly lines has implications for pathway annotation, product prediction, and engineering.

Nature constructs structurally diverse, bioactive molecules using enzymes. Many enzymes catalyze synthetically challenging reactions under mild, physiological conditions. Consequently, they have long been a source of inspiration for developing biomimetic organic syntheses and methods. In addition, enzymes are increasingly being used as biocatalysts in industry. Therefore, the discovery of enzymes that catalyze chemically intriguing transformations can positively impact synthesis in multiple ways. With the recent advances in next-generation DNA sequencing technologies, we are now able to access enormous amount of genomic sequencing data, which encodes a treasure chest of new enzymatic chemistry. The challenge now is to devise a method to efficiently identify chemically interesting enzymes from this vast pool of information.

One possible solution to this problem is to study the biosynthetic pathways of structurally unique natural products, which are predicted to involve novel enzymatic reactions. The cylindrocyclophanes are a family of natural products that contain an unusual [7.7]paracyclophane core scaffold. Based on the previous work on the cylindrocyclophanes, their biosynthesis was predicted to involve an extraordinary C–C bond-formation. To discover the enzyme responsible for this chemistry, we studied the biosynthesis of the cylindrocyclophanes.

Chapter 2 describes the discovery and the validation of the cylindrocyclophane (cyl) biosynthetic gene cluster. The candidate cyl gene cluster was identified from the genomic sequence of the cylindrocyclophane producer. We formulated a biosynthetic hypothesis based on the cyl gene cluster annotation and biochemically characterized the functions of three enzymes (fatty acid activating enzymes CylA/CylB and type III PKS CylI) to connect the cyl gene cluster to the production of the cylindrocyclophanes. In addition, feeding experiments using deuterium-labeled decanoic acid revealed that decanoic acid is a precursor to the cylindrocyclophanes.

Chapter 3 details our investigation of the type I PKS assembly line termination chemistry. We discovered that cylindrocyclophane biosynthesis involves a rare interaction between type I and type III PKSs, in which type III PKS CylI catalyzes assembly line termination by directly using a substrate tethered to the type I PKS CylH. Interestingly, CylH contains a C-terminal TE domain, a domain that catalyzes product release in typical assembly line enzymes. We found that the CylH TE domain has sequence and functional homology to type II editing thioesterases using both bioinformatic analyses and biochemical characterizations.

Chapter 4 details our discovery of a new halogenating enzyme CylC that catalyzes a cryptic chlorination in cylindrocyclophane biosynthesis. Bioinformatic analyses revealed that CylC resembles ferritin-like di-metallo carboxylate enzymes and that CylC and its homologs are likely responsible for chlorination of cyanobacterial natural products. CylC catalyzed chlorination of decanoyl-CylB in vivo, which indicated that cylindrocyclophane biosynthesis requires pre-functionalization of an unactivated carbon center for eventual C–C bond formation.

Chapter 5 discusses our discovery of a new alkylating enzyme CylK that catalyzes a Friedel–Crafts-type C–C bond formation. CylK catalyzed the final dimerization step to construct the [7.7]paracyclophane core of the cylindrocyclophanes using chlorinated resorcinol substrates, confirming that the biosynthesis proceeds through cryptic chlorination. The expanded substrate scope of CylK suggests that this enzyme is a promising candidate for future bioengineering efforts to develop useful biocatalysts for C–C bond formation. Thus, we succeeded in discovering a new halogenase and an alkylating enzyme through our investigation of cylindrocyclophane biosynthesis using a chemically guided approach.

The cylindrocyclophanes are a family of natural products that share a remarkable paracyclophane carbon scaffold. Using genome sequencing and bioinformatic analyses, we have discovered a biosynthetic gene cluster involved in the assembly of cylindrocyclophane F. Through a combination of in vitro enzyme characterization and feeding studies, we have confirmed the connection between this gene cluster and cylindrocyclophane production, elucidated the chemical events involved in initiating and terminating an unusual type I polyketide synthase (PKS) assembly line, and discovered that macrocycle assembly involves functionalization of an unactivated carbon center.