Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

• Published in: Nature communications 2020-06, Vol.11 (1), p.2991-10, Article 2991
• Main Authors: Lukowski, April L., Liu, Jianxin, Bridwell-Rabb, Jennifer, Narayan, Alison R. H.
• Format: Article
• Language: English
• Subjects: 101/58; 631/45/535/1266; 631/92/603; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biocatalysis; Biocatalysts; Biosynthesis; Catalytic Domain; Crystal structure; Cyanobacteria; Cyanobacteria - enzymology; Cyanobacteria - genetics; Divergence; Enzymes; Genetic Variation; Humanities and Social Sciences; Hydroxylation; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Kinetics; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Models, Molecular; multidisciplinary; Mutagenesis; Oxidants; Oxidizing agents; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Protein Conformation; Protein Multimerization; Residues; Saxitoxin; Science; Science (multidisciplinary); Selectivity; Shellfish; Substrate Specificity; Substrates; Toxins
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-16729-0

Biocatalysts that perform C–H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono- or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes. Rieske oxygenases are iron-dependent enzymes that catalyse C–H mono- and dihydroxylation reactions. Here, the authors characterise two cyanobacterial Rieske oxygenases, SxtT and GxtA that are involved in the biosynthesis of paralytic shellfish toxins and determine their substrate free and saxitoxin analog-bound crystal structures and by using mutagenesis experiments identify residues, which are important for substrate positioning and reaction selectivity.