Second, isolating molecules from their solvent and environment allow to study their intrinsic properties. First, because isolated molecules in vacuo cannot exchange energy with their surroundings, reactivity can be studied in well-defined energetic conditions. Importantly, the question of why studying nucleic acids in the gas phase is addressed from three different points of view. Nucleic acid function is also overviewed, from the roles of natural nucleic acids in biology to those of artificial nucleic acids in the biotechnology, biomedical, or nanotechnology fields. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.ĭepartment of Chemistry, University of Adelaide, Adelaide, South Australia 5005, Australia.This introductory chapter sets the stage for the various methods and application that will be described in the book “Nucleic acids in the gas phase.” Using key review articles as references, nucleic acid structures are introduced, with progression from primary structure to the main secondary, tertiary, and quaternary structures of DNA and RNA. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4.
The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions.
Biologically Interesting Molecule Reference Dictionary (BIRD).
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