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

Abstract

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.

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

“…ROs are a family of enzymes best known for catalyzing the NAD(P)H-dependent dihydroxylation of aromatic compounds pollutants ( 10 , 11 ). However, they catalyze a range of oxidation chemistries and have been the focus of extensive investigation, in part because of their potential use in bioremediation and biocatalysis ( 12 , 13 ). RO systems comprise two or three components: an oxygenase and a reductase that transfers electrons from NAD(P)H to the oxygenase, either directly or via a ferredoxin ( 14 ).…”

“…The oxygenase contains a mononuclear iron where catalysis occurs and a Rieske-type iron-sulfur cluster ([2Fe-2S]) that mediates electron transfer to the catalytic center. Structural studies on ROs such as naphthalene dioxygenase (NDO) ( 10 , 15 , 16 , 17 , 18 , 19 ) have identified key features that are responsible for substrate specificity and have provided valuable insight into the catalytic mechanism of these enzymes ( 12 ). For example, a small N-terminal domain harbors the [2Fe-2S] cluster while the catalytic center occurs in a larger C-terminal domain ( 20 ).…”

Design principles for site-selective hydroxylation by a Rieske oxygenase

“…2c). Here, the addition of a hydroxyl group at the C12 α-position allows for the formation of hydrogen bonds with Ser159 and Tyr255, explaining the previously noted structural importance of Tyr255 for selectivity 7 . The remaining GxtA interactions are formed between β-STOH and STX with Asp239 and Gln226 (Fig.…”

“…Currently, two main architectures have been identified within this enzyme class, a hetero-hexamer that contains three β-subunits of unknown function (α 3 β 3 ) and a trimer of the catalytic metallocenter containing α-subunits (α 3 , Fig. 1a, b) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] . In both cases, the active sites are buried deep within the protein interior, a feature shared by many other types of metalloenzymes and more than 60% of proteins [24][25][26][27] .…”

“…Here, the three catalytic α-subunits are shown in gray and the β-subunits are shown in pink, cyan, and purple. b The quaternary architecture of a homotrimeric α 3 Rieske oxygenase is observed in 10 of the 18 available non-redundant Rieske oxygenase crystal structures [7][8][9][10][11][12][13][14] (PDB: 6WN3 7 ). The active site of the α 3 Rieske oxygenases is also buried deep within the protein core.…”