An orally administered enzyme therapeutic for homocystinuria that suppresses homocysteine by metabolizing methionine in the gastrointestinal tract.

Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg-I278T Cbs-/- mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg-I278T Cbs-/- mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU.

Exploring Fungal Polyketide C-Methylation through Combinatorial Domain Swaps.

Polyketide C-methylation occurs during a programmed sequence of dozens of reactions carried out by multidomain polyketide synthases (PKSs). Fungal PKSs perform these reactions iteratively, where a domain may be exposed to and act upon multiple enzyme-tethered intermediates during biosynthesis. We surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs to gain insight into how different methylation patterns are installed. Our in vitro results show that control of methylation resides primarily with the CMeT, and CMeTs can intercept and methylate intermediates from noncognate nonreducing PKS domains. Furthermore, the methylation pattern is likely imposed by a competition between methylation or ketosynthase-catalyzed extension for each intermediate. Understanding site-specific polyketide C-methylation may facilitate targeted C-C bond formation in engineered biosynthetic pathways.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

The structural organization of substrate loading in iterative polyketide synthases.

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.

Chromobacterium spp. mediate their anti-Plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin.

The Chromobacterium sp. Panama bacterium has in vivo and in vitro anti-Plasmodium properties. To assess the nature of the Chromobacterium-produced anti-Plasmodium factors, chemical partition was conducted by bioassay-guided fractionation where different fractions were assayed for activity against asexual stages of P. falciparum. The isolated compounds were further partitioned by reversed-phase FPLC followed by size-exclusion chromatography; high resolution UPLC and ESI/MS data were then collected and revealed that the most active fraction contained a cyclic depsipeptide, which was identified as romidepsin. A pure sample of this FDA-approved HDAC inhibitor allowed us to independently verify this finding, and establish that romidepsin also has potent effect against mosquito stages of the parasite's life cycle. Genomic comparisons between C. sp. Panama and multiple species within the Chromobacterium genus further demonstrated a correlation between presence of the gene cluster responsible for romidepsin production and effective antiplasmodial activity. A romidepsin-null Chromobacterium spp. mutant loses its anti-Plasmodium properties by losing the ability to inhibit P. falciparum HDAC activity, and romidepsin is active against resistant parasites to commonly deployed antimalarials. This independent mode of action substantiates exploring a chromobacteria-based approach for malaria transmission-blocking.

Starter unit flexibility for engineered product synthesis by the nonreducing polyketide synthase PksA.

Nonreducing polyketide synthases (NR-PKSs) are unique among PKSs in their domain structure, notably including a starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain that selects an acyl group as the primer for biosynthesis, most commonly acetyl-CoA from central metabolism. This clan of mega-enzymes resembles fatty acid synthases (FASs) by sharing both their central chain elongation steps and their capacity for iterative catalysis. In this mode of synthesis, catalytic domains involved in chain extension exhibit substrate plasticity to accommodate growing chains as small as two carbons to 20 or more. PksA is the NR-PKS central to the biosynthesis of the mycotoxin aflatoxin B1 whose SAT domain accepts an unusual hexanoyl starter from a dedicated yeast-like FAS. Explored in this paper is the ability of PksA to utilize a selection of potential starter units as substrates to initiate and sustain extension and cyclization to on-target, programmed polyketide synthesis. Most of these starter units were successfully accepted and properly processed by PksA to achieve biosynthesis of the predicted naphthopyrone product. Analysis of the on-target and derailment products revealed trends of tolerance by individual PksA domains to alternative starter units. In addition, natural and un-natural variants of the active site cysteine were examined and found to be capable of biosynthesis, suggesting possible direct loading of starter units onto the ß-ketoacyl synthase (KS) domain. In light of the data assembled here, the predictable synthesis of unnatural products by NR-PKSs is more fully defined.