Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Abstract

Members of the antibiotic biosynthesis monooxygenase family catalyze O 2 -dependent oxidations and oxygenations in the absence of any metallo-or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O 2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater. As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pK a . The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O 2 -dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O 2 reactivity via markedly different means.

Catalytic Mechanism of Nogalamycin Monoxygenase: How Does Nature Synthesize Antibiotics without a Metal Cofactor?

“…Experimental work above the singleprotonation pK a of the enzyme-substrate complex (6.9) showed that oxygenation is the dominant process, whereas in acidic environments the reaction appeared to undergo a net one-electron/one-proton oxidation to yield a dimeric product. 33 The latter is an unwanted side product in the biological context, as it is off the pathway leading ultimately to the antibiotic. As such, the reaction processes for electron transfer, hydrogen atom abstraction and hydride transfer were repeated using SubHas the substrate (right-hand-side of Figure 3).…”

“…Our work contrasts experimental suggestions that upon O 2 binding a spontaneous electron transfer takes place. 32,33 In recent QM/MM calculations on the firefly luciferin reaction it was found that O 2 upon binding nearby a luciferin-adenylate led to spontaneous electron transfer. 65 Clearly, the ionization potential of SubH 2 used here is considerably different from luciferin-adenylate and consequently no electron transfer is possible.…”

“…Nogalamycin monoxygenase (NMO) is an antibiotic biosynthesis monoxygenase from Staphylococcus nogalater that catalyzes the conversion of 12-deoxy-nogalonic acid to nogalonic acid ( Figure 1). 32,33 A 1.9Å resolution crystal structure has been measured for the enzyme with a molecule of ethylene glycol bound, which has been replaced by a model of the native substrate in the left-hand-side of Figure 1. [34][35][36][37] The substrate pocket is lined with aromatic amino acid residues, e.g., Phe 16 , Phe 20 , Phe 73 and Trp 67 , which may have functions related to either -stacking with the substrate or electron transfer.…”

Rubisco is not really so bad

“…Hence, it has been proposed that so called "cofactor-independent" oxygenases proceed via a "substrate-assisted" mechanism, whereby the depronated substrate (carbanion) is able to directly transfer an electron to O 2 (as flavin cofactors do; Massey, 1994), leading to the formation of a substrate radical pair that recombines into a peroxide (Bui & Steiner, 2016;Machovina, Usselman, & DuBois, 2016;Silva, 2016 , 2015). Regardless of mechanistic details, the flavin analogy is very interesting, because the reactivity of protein-bound flavins towards O 2 has been shown to vary widely across flavoproteins, depending on their class and substrate specificity, with no obvious relation to their one electron redox potential E 0 ' (Mattevi, 2006).…”

Refining the reaction mechanism of O2towards its co-substrate in cofactor-free dioxygenases

“…It thus appears that for all cofactor-less oxidases studied computationally so far (urate oxidase, 1- H -3-hydroxy-4-oxoquinaldine-2,4-dioxygenase, coproporphyrinogen oxidase and vitamin K-dependent glutamate carboxylase) catalysis occurs through direct electron transfer from substrate to O 2 followed by radical recombination, instead of minimum-energy crossing points without formal electron transfer. A novel nogalamycin monooxygenase (Machovina, Usselman & DuBois, 2016) has recently been shown experimentally to also follow this paradigm, which thus appears to be the most general strategy for the enzyme-catalyzed reaction of triplet O 2 with conjugated organic substrates in the absence of metal cofactors.…”