Abstract
The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.
Highlights
The flavoenzyme morphinone reductase (MR)1 from Pseudomonas putida is a member of the Old Yellow Enzyme (OYE) family of proteins [1]
The overall subunit structure is similar to that reported for OYE [21] and pentaerythritol tetranitrate (PETN) reductase [22]
-strands A and B form a cap at the N-terminal domain of the barrel, as seen in OYE [21] and PETN reductase [22]
Summary
Wavelength (Å) Total reflections Unique reflections Cell (Å) Resolution (Å) Completeness (%) Rmerge (%), outer shell T/ (T). Ability to reduce the unsaturated carbon-carbon bond of a number of opiate compounds [27]. Sequence alignment studies suggest differences in the chemical nature of the active site of MR compared with OYE and PETN reductase [1]. To investigate these aspects, and to provide a structural basis for detailed mechanistic studies of MR, we have solved the structure of MR in complex with the opiate substrate codeinone at 2.2-Å resolution. The structure is discussed in the context of the mechanism of opiate reduction and in view of the existing structural information for PETN reductase and OYE.
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