A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase

Abstract

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90°rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137 of the neighbouring protomers. To investigate the importance of the ion pair contacts, the K109L and D137L single replacement variants, as well as the K109L ⁄ D137L and K109D ⁄ D137K double replacement variants, were generated, expressed, purified, crystallized and biochemically characterized. The K109L and D137L variants form dimers instead of tetramers, whereas the K109L ⁄ D137L and K109D ⁄ D137K variants appear to exist in a dimer-tetramer equilibrium in solution. The crystal structures of the K109L and D137L variants confirm the dimeric state, with the K109L ⁄ D137L and K109D ⁄ D137K variants adopting a tetrameric assembly. Interestingly, all protein variants show a drastically reduced quinone reductase activity in steady-state kinetics. Detailed analysis of the two half reactions revealed that the oxidative half reaction is not affected, whereas reduction of the bound FMN cofactor by NADPH is virtually abolished. Inspection of the crystal structures indicates that the side chain of K109 plays a dual role by forming a salt bridge to D137, as well as stabilizing a glycine-rich loop in the vicinity of the FMN cofactor. In all protein variants, this glycine-rich loop exhibits a much higher mobility, compared to the wild-type. This appears to be incompatible with NADPH binding and thus leads to abrogation of flavin reduction. Abbreviations DSC, differential scanning calorimetry; DLS, dynamic light scattering; Ni-NTA, nickel-nitrilotriacetic acid agarose.

Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR

“…The homolog of SAV2522 in B. subtilis yfiE indeed possesses metabolic activity of catechol degradation (43). In addition, SAV0340, which encodes a NADH-dependent FMN reductase, could act on the detoxification of quinones (44,45); its homolog yhdA in B. subtilis functions as a reductase for a variety of quinone molecules (46). Meanwhile, a nitroreductase, encoded by SAV2033, may contribute to a two-electron reduction of nitro groups in nitroaromatic compounds as well as quinones (47,48).…”

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…ecAzoR, rsAzoR, stAzoR and efAzoR are azoreductases from E. coli (Nakanishi et al, 2001), R. sphaeroides (Bin et al, 2004), S. typhimurium (Zhang et al, 2004) and E. faecalis (Chen et al, 2004). paWrbA is the sequence for WrbA from P. aeruginosa (Gorman and Shapiro, 2005), KefF is an enzyme from E. coli (Roosild et al, 2009), ecMdaB and paMdaB are the mediator of drug activity B from E. coli (Adams and Jia, 2006) and P. aeruginosa respectively, YhdA is an enzyme from B. subtilis (Binter et al, 2009), EmoB is an enzyme fromMesorhizobiums p. BNC1 (Nissen et al, 2008), ArsH is an enzyme from S. meliloti (Ye et al, 2007), Lot6p is an enzyme from S. cerevisiae (Liger et al, 2004), ppChrR is the chromate reductase from P. putida (Ackerley et al, 2004). 1X77 is an NADPH dependent oxidoreductase from P. aeruginosa (Agarwal et al, 2006), hNQO1 and hNQO2 are human NQOs (Foster et al, 1999;Li et al, 1995), A. tha is the sequence for an NAD(P)H quinone reductase from A. thaliana (Sparla et al, 1999).…”

“…When Tyr131 was mutated to Phe within the active site of paAzoR1 (equivalent residues are prolines in paAzoR2 and paAzoR3), it caused a significant shift in T m to approximately 42°C (Wang et al, 2010a). Oligomerisation state has previously been suggested as a possible reason for high thermostability (Deller et al, 2006), although later work on B. subtilis azoreductase disagreed (Binter et al, 2009). Preliminary data from analytical ultracentrifugation experiments on paAzoR2 and paAzoR3 (Wang, 2008) indicate they form dimeric and trimeric (Ryan et al, 2010)).…”

“…Despite some of them sharing only minimal sequence identity, they have a common three dimensional fold (Fig. 1B) (Li et al, 1995;Ito et al, 2006;Wang et al, 2007;Ye et al, 2007;Binter et al, 2009).…”

“…A number of bacterial enzymes, classified as azoreductases, can also reduce quinones (Nakanishi et al, 2001;Chen et al, 2004;Liu et al, 2008;Binter et al, 2009). The mechanism for azo reduction that has been proposed (Ryan et al, 2010) explains the ability of azoreductases to reduce both azo and quinone substrates.…”