5tz5 | pdb_00005tz5

Crystal Structure of CurK Dehydratase H996F Inactive Mutant

spinqualitylabelsall models 5tz5, resolution 1.43Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5tz5 is a 2 chain structure with sequence from Moorena producens 3L. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.428Å Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function F4Y425_9CYAN Publication Abstract from PubMed Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels, and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates, and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all-trans-trienoate-forming CurK-DH, whose kcat of 72 s-1 is more than 3 orders of magnitude greater than that of any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH DHs on the basis of incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ-DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology. Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis.,Fiers WD, Dodge GJ, Sherman DH, Smith JL, Aldrich CC J Am Chem Soc. 2016 Dec 14;138(49):16024-16036. Epub 2016 Nov 30. PMID:27960309 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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