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Subunit Interactions in the FAD‐Exchange Mechanism of Styrene Monooxygenase
Author(s) -
Chen Patrick,
Gassner George
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.527.2
Subject(s) - chemistry , enantiopure drug , monooxygenase , protein subunit , cofactor , flavin group , flavoprotein , biocatalysis , active site , stereochemistry , styrene , catalytic cycle , combinatorial chemistry , catalysis , biochemistry , enzyme , reaction mechanism , organic chemistry , copolymer , enantioselective synthesis , polymer , cytochrome p450 , gene
Styrene monooxygenase (SMO), a FAD‐dependent two‐component flavoprotein composed of reductase (SMOB) and FAD‐specific styrene epoxidase (SMOA), is targeted as a biocatalyst due to its ability to form enantiopure epoxides from a range of styrene derivatives. SMO can produce chiral oxides in 99% enantiomeric excess in the aqueous phase and outperforms the best currently available organic and organometallic synthetic catalysts in reaction rate and efficiency. One of the outstanding challenges encountered in the development of SMO as scalable biocatalysis is the two‐component structure of this enzyme and the associated requirement of coenzyme recycling. Several approaches have been evaluated to optimize reaction conditions including use of surrogates for the flavin and pyridine nucleotide coenzymes required in these reactions. Each of these steps has contributed to the development of SMO and related flavin monooxygenases as biocatalysts, but the nature of the protein‐protein and protein‐subunit interactions has remained elusive. 4‐hydroxybenzoate‐3‐hydroxylase (PDB id: 1PBE), a similar structure to SMO, reveals an alternative subunit interface (FAD‐exchange state) that could provide SMOB direct access to the active site of SMOA. We propose that in catalysis the binding energy of reduced FAD is coupled to the conversion of SMOA from apo‐resting state to an FAD‐exchange state. Our study sought to understand the correlation between FAD‐binding and the conformational change of SMOA during catalysis. To evaluate this hypothesis of alternate subunit interfaces in catalysis, structure files of SMOA subunits were submitted to the ClusPro 2.0 protein interactions server. Inspection of the resulting models indicate that lysine residues sequestered in the interface of the resting‐state subunits are exposed in the exchange‐state. Based on modeling studies different lysine residues are hypothesized to be sequestered at the dimer interface of the resting and FAD‐exchange states of SMOA. These lysines were targeted for labeling with fluorescent probes dansyl‐Cl (5‐ (dimethylamino)naphthalene‐1‐sulfonyl chloride), FITC (fluorescein isothiocyanate), and RITC (rhodamine isothiocyanate). By reacting SMOA with dansyl‐Cl after reduction of SMOA in 50% glycerol in the presence of 100 μM styrene, we were able to trap SMOA in a configuration related to the exchange state. After labeling SMOA(FAD red ) with Dansyl‐Cl under anaerobic conditions, followed by air‐reoxidation, the enzyme retains activity, but binds tightly to FAD ox . This feature is consistent with the FAD‐exchange state of SMOA, which is postulated to bind FAD ox with high affinity relative to the resting state. A structural model of the FAD‐exchange complex supported by steady‐state fluorescence anisotropy, fluorescence lifetime measurements, and analytical gel filtration data will be presented. Support or Funding Information Supported by the NIH MARC T34‐GM008574, DRC (Development of Research and Creativity) Grant San Francisco State University. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .