3hrr
The Product Template Domain from PksA with Harris Compound Bound
Structural highlights
FunctionAFLC_ASPPU Norsolorinic acid synthase; part of the gene cluster that mediates the biosynthesis of aflatoxins, a group of polyketide-derived furanocoumarins, and part of the most toxic and carcinogenic compounds among the known mycotoxins (PubMed:7592391, PubMed:15094053, PubMed:7565588, PubMed:15006741, PubMed:17086560, PubMed:18403714). The four major aflatoxins produced by A.parasiticus are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) (PubMed:15006741). Within the aflatoxin pathway, the norsolorinic acid synthase aflC combines a hexanoyl starter unit provided to the acyl-carrier protein (ACP) domain by the fungal fatty acid synthase aflA/aflB, and 7 malonyl-CoA extender units to synthesize the precursor norsolorinic acid (NOR) (PubMed:17086560, PubMed:18403714). The biosynthesis of aflatoxins begins with the norsolorinic acid synthase aflC that combines a hexanoyl starter unit produced by the fatty acid synthase aflA/aflB and 7 malonyl-CoA extender units to synthesize the precursor NOR. The second step is the conversion of NOR to averantin (AVN) and requires the norsolorinic acid ketoreductase aflD, which catalyzes the dehydration of norsolorinic acid to form (1'S)-averantin. The norsolorinic acid reductases aflE and aflF may also play a role in the conversion of NOR to AVN. The cytochrome P450 monooxygenase aflG then catalyzes the hydroxylation of AVN to 5'hydroxyaverantin (HAVN). The next step is performed by the 5'-hydroxyaverantin dehydrogenase aflH that transforms HAVN to 5'-oxoaverantin (OAVN) which is further converted to averufin (AVF) by aflK that plays a dual role in the pathway, as a 5'-oxoaverantin cyclase that mediates conversion of 5'-oxoaverantin, as well as a versicolorin B synthase in a later step in the pathway. The averufin oxidase aflI catalyzes the conversion of AVF to versiconal hemiacetal acetate (VHA). VHA is then the substrate for the versiconal hemiacetal acetate esterase aflJ to yield versiconal (VAL). Versicolorin B synthase aflK then converts VAL to versicolorin B (VERB) by closing the bisfuran ring of aflatoxin which is required for DNA-binding, thus giving to aflatoxin its activity as a mutagen. Then, the activity of the versicolorin B desaturase aflL leads to versicolorin A (VERA). A branch point starts from VERB since it can also be converted to dihydrodemethylsterigmatocystin (DMDHST), probably also by aflL, VERA being a precursor for aflatoxins B1 and G1, and DMDHST for aflatoxins B2 and G2. Next, the versicolorin reductase aflM and the cytochrome P450 monooxygenase aflN are involved in conversion of VERA to demethylsterigmatocystin (DMST). AflX and aflY seem also involved in this step, through probable aflX-mediated epoxide ring-opening step following versicolorin A oxidation and aflY-mediated Baeyer-Villiger oxidation required for the formation of the xanthone ring. The methyltransferase aflO then leads to the modification of DMST to sterigmatocystin (ST), and of DMDHST to dihydrosterigmatocystin (DHST). Both ST and DHST are then substrates of the O-methyltransferase aflP to yield O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST), respectively. Finally OMST is converted to aflatoxins B1 and G1, and DHOMST to aflatoxins B2 and G2, via the action of several enzymes including O-methylsterigmatocystin oxidoreductase aflQ, the cytochrome P450 monooxygenase aflU, but also the NADH-dependent flavin oxidoreductase nadA which is specifically required for the synthesis of AFG1 (PubMed:15006741).[1] [2] [3] [4] [5] [6] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedPolyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-beta-keto intermediates. How poly-beta-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 A crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B(1) in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization.,Crawford JM, Korman TP, Labonte JW, Vagstad AL, Hill EA, Kamari-Bidkorpeh O, Tsai SC, Townsend CA Nature. 2009 Oct 22;461(7267):1139-43. PMID:19847268[7] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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