Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase fromEscherichia coli

Abstract

The alkanesulfonate FMN reductase (SsuE) from Escherichia coli catalyzes the reduction of FMN by NADPH to provide reduced flavin for the monooxygenase (SsuD) enzyme. The vapor-diffusion technique yielded single crystals that grow as hexagonal rods and diffract to 2.9 A resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. The SsuE protein lacks any cysteine or methionine residues owing to the role of the SsuE enzyme in the acquisition of sulfur during sulfate starvation. Therefore, substitution of two leucine residues (Leu114 and Leu165) to methionine was performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN. These preliminary studies should lead to the structure solution of SsuE. It is anticipated that this new protein structure will provide detailed structural information on specific active-site regions of the protein and insight into the mechanism of flavin reduction and transfer of reduced flavin.

Crystal Structure of Escherichia coli SsuE: Defining a General Catalytic Cycle for FMN Reductases of the Flavodoxin-like Superfamily

“…The lack of FMNH 2 binding in chain B may be explained in that Arg10 makes a crystal contact (with Ser141 of chain C′) and this stabilizes the “out” conformation of Arg10 as seen in its temperature factors being lower than those of the other chains. Apparently, the weaker binding of FMNH 2 ( K d values of 15.5 and 0.015 μM for FMNH 2 and FMN, respectively) , is not strong enough to cause Arg10 to shift, which happens upon FMN binding. Interestingly, in chains A and C, Arg10 has partial occupancy of the “in” and “out” conformations modeled at 0.67 and 0.33, respectively.…”

“…Overall Structure. Recombinant SsuE crystallized readily in space group P6 1 22 as previously reported, 18 and we determined its structure by molecular replacement using the EmoB crystal structure (37% sequence identity). Structures with reasonable R factors at resolutions near 2 Å (Table 1) were determined in three states: apo-SsuE as grown, an FMN-bound form resulting from a 1 mM FMN soak, and an FMNH 2 -bound form resulting from dithionite treatment of FMN-soaked crystals.…”

“…For crystallization, SsuE was concentrated to ∼10 mg/ mL in 10 mM HEPES (pH 7.0). Building on previous work, 18 we grew crystals at room temperature using hanging drops made with 4 μL of a protein stock mixed with 2 μL of a reservoir containing 7.5% (w/v) poly(ethylene glycol) (PEG) 3350 and 0.1 M sodium citrate. For storage and soaks, crystals were transferred to an artificial mother liquor (AML) like the reservoir but with 20% (w/v) PEG 3350.…”

In silico screening and heterologous expression of soluble dimethyl sulfide monooxygenases of microbial origin in Escherichia coli

“…Flavin reductases associated with monooxygenases are classified into two classes. Class I enzymes contain bound flavin and are referred as standard flavoprotein, e.g., the sulfite reductase (Fre) from E. coli and Frp reductase from Vibrio harveyi (Coves et al 1993 ; Lei et al 1994 ); class II enzymes do not contain any bound flavin groups and are defined as non-standard flavoproteins, e.g., SsuE from E. coli (Gao et al 2005 ).…”