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Protein Sci. 2019 Jan;28(1):123-134. doi: 10.1002/pro.3504. Epub 2018 Nov 15.

Not as easy as π: An insertional residue does not explain the π-helix gain-of-function in two-component FMN reductases.

Author information

1
The Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, 66045.
2
The Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama, 36849.

Abstract

The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix. The structure of the Y118A SsuE π-helix was converted to an α-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the π-helix was altered, the FMN binding region remained unchanged. Conversely, deletion of Tyr118 disrupted the secondary structural properties of the π-helix, generating a random coil region in the middle of helix 4. Both the Y118A and Δ118 SsuE SsuE variants crystallize as a dimer. The MsuE FMN reductase involved in the desulfonation of methanesulfonates is structurally similar to SsuE, but the π-helix contains a His insertional residue. Exchanging the π-helix insertional residue of each enzyme did not result in equivalent kinetic properties. Structure-based sequence analysis further demonstrated the presence of a similar Tyr residue in an FMN-bound reductase in the NADPH:FMN reductase family that is not sufficient to generate a π-helix. Results from the structural and functional studies of the FMN-dependent reductases suggest that the insertional residue alone is not solely responsible for generating the π-helix, and additional structural adaptions occur to provide the altered gain of function.

KEYWORDS:

MsuD; MsuE; NAD(P)H-FMN reductase family; SsuD; SsuE; flavin monooxygenases; flavin reductases; two-component FMN-dependent systems; π-helix

PMID:
30171650
PMCID:
PMC6295896
[Available on 2020-01-01]
DOI:
10.1002/pro.3504

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