Abstract

Es müssen nicht immer Thioester sein: Cryptophycin-Thioesterase (Crp TE) spaltet lineare Cryptophycine, die an aktivierte PEGA-Harze gebunden sind, und überführt sie in Makrolactone. Diese neuartige enzymatische Umwandlung an der Festphase wurde verwendet, um die Toleranz von Crp TE in Bezug auf Änderungen in der Substratstruktur zu untersuchen.

Abstract

An unexpected biochemical strategy for chain initiation is described for the loading module of the polyketide synthase of curacin A, an anticancer lead derived from the marine cyanobacterium Lyngbya majuscula. A central GCN5-related N-acetyltransferase (GNAT) domain bears bifunctional decarboxylase/S-acetyltransferase activity, both unprecedented for the GNAT superfamily. A CurA loading tridomain, consisting of an adaptor domain, the GNAT domain, and an acyl carrier protein, was assessed biochemically, revealing that a domain showing homology to GNAT (GNAT_L) catalyzes (i) decarboxylation of malonyl-coenzyme A (malonyl-CoA) to acetyl-CoA and (ii) direct S-acetyl transfer from acetyl-CoA to load an adjacent acyl carrier protein domain (ACP_L). Moreover, the N-terminal adapter domain was shown to facilitate acetyl-group transfer. Crystal structures of GNAT_L were solved at 1.95 angstroms (ligand-free form) and 2.75 angstroms (acyl-CoA complex), showing distinct substrate tunnels for acyl-CoA and holo-ACP_L binding. Modeling and site-directed mutagenesis experiments demonstrated that histidine-389 and threonine-355, at the convergence of the CoA and ACP tunnels, participate in malonyl-CoA decarboxylation but not in acetyl-group transfer. Decarboxylation precedes acetyl-group transfer, leading to acetyl-ACP_L as the key curacin A starter unit.

Thioesters need not apply: Cryptophycin thioesterase (Crp TE) cleaves and macrolactonizes linear cryptophycin substrates bound to activated safety-catch PEGA resin. This novel enzymatic solid-phase approach was used to further investigate the tolerance of Crp TE for structural variations of substrates.

More pieces for the puzzle. The β-methylaspartate-α-decarboxylase (CrpG) from the cryptophycin biosynthetic pathway was cloned, over-expressed, and purified. We found that CrpG decarboxylates (2S,3R)-3-methylaspartic acid to form 3-amino-2(R)-methylpropionic acid, which is subsequently incorporated into Unit C of cryptophycins (see scheme).

Marine organisms are a rich source of secondary metabolites. They have yielded thousands of compounds with a broad range of biomedical applications. Thus far, samples required for preclinical and clinical studies have been obtained by collection from the wild, by mariculture, and by total chemical synthesis. However, for a number of complex marine metabolites, none of these options is feasible for either economic or environmental reasons. In order to proceed with the development of many of these promising therapeutic compounds, a reliable and renewable source must be found. Over the last twenty years, the study of microbial secondary metabolites has greatly advanced our understanding of how nature utilizes simple starting materials to yield complex small molecules. Much of this work has focused on polyketides and nonribosomal peptides, two classes of molecules that are prevalent in marine micro- and macroorganisms. The lessons learned from the study of terrestrial metabolite biosynthesis are now being applied to the marine world. As techniques for cloning and heterologous expression of biosynthetic pathways continue to improve, they may provide our greatest hope for bridging the gap between the promise and application of many marine natural products.