8erj | pdb_00008erj
Crystal structure of Fub7 in complex with E-2-aminocrotonate
Structural highlights
FunctionFUB7_GIBF5 Sulfhydrylase; part of the gene cluster that mediates the biosynthesis of fusaric acid, a mycotoxin with low to moderate toxicity to animals and humans, but with high phytotoxic properties (PubMed:26662839). L-aspartate is suggested as fusaric acid amino acid precursor that is activated and further processed to O-acetyl-L-homoserine by cluster enzymes aspartate kinase FUB3 and homoserine O-acetyltransferase FUB5, as well as enzymes of the primary metabolism (PubMed:26662839). The polyketide synthase (PKS) FUB1 generates the triketide trans-2-hexenal which is presumptively released by the hydrolase FUB4 and linked to the NRPS-bound amino acid precursor by NAD(P)-dependent dehydrogenase FUB6 (PubMed:26662839). FUB1, FUB4, and the non-canonical NRPS Fub8 may form an enzyme complex (PubMed:26662839). Further processing of the NRPS-bound intermediate might be carried out by FUB6 and the O-acetylhomoserine FUB7, enabling a spontaneous electrocyclization to close the carbon backbone of fusaric acid (PubMed:26662839). Dihydrofusaric acid is likely to be released via reduction by the thioester reductase (TR) domain of FUB8 whereupon the final oxidation to fusaric acid may (also) be performed by the FMN-dependent dehydrogenase FUB9 (PubMed:26662839).[1] Publication Abstract from PubMedPyridoxal 5'-phosphate (PLP)-dependent enzymes that catalyze gamma-replacement reactions are prevalent, yet their utilization of carbon nucleophile substrates is rare. The recent discovery of two PLP-dependent enzymes, CndF and Fub7, has unveiled unique C-C bond forming capabilities, enabling the biocatalytic synthesis of alkyl- substituted pipecolic acids from O-acetyl-L-homoserine and beta-keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of pipecolic acid derivatives. However, the catalytic mechanisms of these enzymes remain elusive, and a dearth of structural information hampers their extensive application. Here, we have broadened the catalytic scope of Fub7 by employing ketone-derived enolates as carbon nucleophiles, revealing Fub7's capacity for substrate-dependent regioselective alpha-alkylation of unsymmetrical ketones. Through an integrated approach combining X-ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer a detailed mechanistic insight into Fub7 catalysis. Our findings elucidate the structural basis for its substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods to access a broader array of noncanonical amino acids. Molecular and Structural Basis for Cgamma-C Bond Formation by PLP-Dependent Enzyme Fub7.,Liu S, Yeh C, Reavill C, Jones B, Zou Y, Hai Y Angew Chem Int Ed Engl. 2024 Mar 22;63(13):e202317161. doi: , 10.1002/anie.202317161. Epub 2024 Feb 20. PMID:38308582[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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