Abstract
How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.
Highlights
How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level
Using Y183F-xenobiotic reductase A (XenA), we prepared stable, yet minimally perturbed Michaelis complexes, which we investigated using X-ray crystallography, stopped-flow kinetics and resonance Raman (RR) spectroscopy
We showed previously that the Y183F-substitution Y183 slows down the reoxidation of reduced XenA with 2-cyclohexenone by two orders of magnitude[7]
Summary
How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. To structurally investigate Michaelis complexes, the native substrate is often replaced by a less reactive analogue or competitive inhibitor, or an inactive state of the enzyme is used[1,2] The latter approach is straightforward for cofactor-dependent enzymes catalysing redox reactions, because they are typically active in only one specific oxidation state. In the case of flavin-dependent oxidoreductases, the oxidized state of the cofactor, which is unable to provide electrons to reduce the substrate, is used to produce non-reactive and stable enzyme–substrate complexes mimicking the Michaelis complex (Michaelis mimic). We have employed xenobiotic reductase A (XenA) from Pseudomonas putida 86, a versatile catalyst belonging to the old yellow enzyme (OYE) family of flavoproteins Enzymes of this family catalyse the reduction of the double bond of a wide range of a,b-unsaturated carbonyl compounds, making them attractive for biotechnological applications. The combined study provides direct evidence for oxidation-state-dependent substrate–cofactor interactions and shows that cofactor compression creates a strong donor–acceptor complex between reduced flavin and the substrates
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