Protein Cell 2010, 1(8): 780–790 Protein & Cell DOI 10.1007/s13238-010-0090-2 RESEARCH ARTICLE Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases ✉ Ali Ryan*, Chan-Ju Wang*, Nicola Laurieri*, Isaac Westwood, Edith Sim Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK ✉ Correspondence: [email protected] Received June 2, 2010 Accepted June 28, 2010 ABSTRACT the dye used for dying fabrics does not bind to the fabric, and as a result, is lost in the effluent (Shore, 1995). As these dyes Azoreductases are involved in the bioremediation by can be carcinogenic (Alves de Lima et al., 2007) their removal bacteria of azo dyes found in waste water. In the gut flora, from the effluent is essential. This has driven investigations they activate azo pro-drugs, which are used for treatment into both electrochemical reduction of azo dyes (Wang et al., of inflammatory bowel disease, releasing the active 2010b), as well as the use of both aerobic and anaerobic component 5-aminosalycilic acid. The bacterium bacteria for bioremediation (dos Santos et al., 2007; You et P. aeruginosa has three azoreductase genes, paAzoR1, al., 2007). paAzoR2 and paAzoR3, which as recombinant enzymes Azo bonds are also found in some drugs including those have been shown to have different substrate specifici- used for the treatment of inflammatory bowel disease (IBD). ties. The mechanism of azoreduction relies upon tauto- Azo compounds have also been shown to be effective merisation of the substrate to the hydrazone form. We antibacterial (Farghaly and Abdalla, 2009) and antitumor (El- report here the characterization of the P. aeruginosa Shafei et al., 2009) agents. Azo drugs for the treatment of IBD azoreductase enzymes, including determining their ther- consist of a carrier compound linked via an azo bond to 5- mostability, cofactor preference and kinetic constants amino salycilic acid (5-ASA). 5-ASA if administered orally is against a range of their favoured substrates. The rapidly removed from the digestive system and so it is expression levels of these enzymes during growth of modified with an inert carrier to prolong its action (Lichtenstein P. aeruginosa are altered by the presence of azo and Kamm, 2008). The azo bond is cleaved by intestinal substrates. It is shown that enzymes that were originally bacteria (Peppercorn and Goldman, 1972) releasing 5-ASA. described as azoreductases, are likely to act as NADH Enzymes able to reduce an azo bond have been found in a quinone oxidoreductases. The low sequence identities number of species including Escherichia coli (Nakanishi et al., observed among NAD(P)H quinone oxidoreductase and 2001), Pseudomonas aeruginosa (Wang et al., 2007), azoreductase enzymes suggests convergent evolution. Enterococcus faecalis (Chen et al., 2004), Sinorhizobium meliloti (Ye et al., 2007), Saccharomyces cerevisiae (Sollner KEYWORDS azoreductase, enzyme mechanism, et al., 2007) and humans (Cui et al., 1995). These enzymes NAD(P)H quinone oxidoreductase, enzyme characterisation, are flavin dependent oxidoreductases. Despite some of them convergent evolution sharing only minimal sequence identity, they have a common three dimensional fold (Fig. 1B) (Li et al., 1995; Ito et al., 2006; INTRODUCTION Wang et al., 2007; Ye et al., 2007; Binter et al., 2009). The azoreductase reaction is proposed to be catalyzed via Dyes that contain an azo bond (−N=N−) contribute sig- a Bi-Bi ping-pong mechanism (Nakanishi et al., 2001; Liu nificantly to world dye production (Stolz, 2001). Almost half of et al., 2008; Wang et al., 2010a), and we have indicated that the reaction is likely to occur via reduction of the hydrazone *These authors contributed equally to the work. 780 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 Azoreductase mechanism Protein & Cell tautomer of the substrate, as identified from structural studies a preference for NADPH (Wang et al., 2007). Interestingly, (Fig. 1A) (Ryan et al., 2010). The first reduction reaction is this preference was reversed on mutation of Tyr131 to proposed to be followed by cleavage of the hydrazine phenylalanine in the enzyme’s active site (Wang et al., intermediate via an acid or base catalyzed reaction (Fig. 1C). 2010a). In contrast to paAzoR1, which shows a preference for This cleavage releases a quinoneimine that is believed to be NADPH as the hydride donor, both paAzoR2 and paAzoR3 the substrate for the second reduction reaction (Fig. 1D). show a preference for NADH (Fig. 3A). paAzoR1 shows the A number of bacterial enzymes, classified as azoreduc- same hydride donor preference when reducing the quinonei- tases, can also reduce quinones (Nakanishi et al., 2001; mine 2,6-dichloroindophenol (DCIP) as it does when reducing Chen et al., 2004; Liu et al., 2008; Binter et al., 2009). The the azo substrate methyl red (Fig. 3B); however, the mechanism for azo reduction that has been proposed (Ryan difference in quinone reductase activity with each of the two et al., 2010) explains the ability of azoreductases to reduce nicotinamide cofactors is smaller than what is observed when both azo and quinone substrates. It has been suggested that reducing azo substrates. In contrast paAzoR3 utilizes both the major biological role of azoreductases is in the detoxifica- NADH and NADPH with a preference for NADH of less than tion of quinones (Liu et al., 2008, 2009). These data suggest twofold when reducing methyl red, while the rate of reduction that azoreductases should be reclassified as NAD(P)H of DCIP with NADH is almost 20 times greater than with quinone oxidoreductases (NQOs). NQOs have also been NADPH (Fig. 3C). shown to be important for bacterial metabolism of some The reasons behind the nicotinamide cofactor preference substrates, e.g., p-nitrophenol (Zhang et al., 2009). Even remain unclear. Two crystal structures have been solved though mammalian NQOs have very low sequence identity showing nicotinamide cofactors bound to flavodoxin-like with the bacterial enzymes (less than 15% between paAzoR1 proteins. The first structure showed rat NQO binding and either human NQO1 or NQO2), they also reduce quinone NADP+ and has been withdrawn from the PDB (Li et al., and azo compounds (Cui et al., 1995). Recent evidence 1995). The second structure shows NADH binding to EmoB implicates the eukaryotic NQOs in the regulation of protea- from Mesorhizobium sp. BNC1 (PDB: 2VZJ) (Nissen et al., somal degradation of tumor suppressor proteins (Sollner et 2008). NADH binds EmoB in a solvent exposed cleft that al., 2009b; Alard et al., 2010). Therefore, further under- extends from the solvent channel occupied by methyl red in standing of the relationship between azoreductases and the structure bound to paAzoR1. When the structure of EmoB NAD(P)H quinone oxidoreductases has wide implications in is aligned with the paAzoR1, however, there are significant biology. clashes between residue side chains and NADH; as a result, Three azoreductases have been identified in the genome it is unlikely that NADH will bind in this cleft. The most possible of P. aeruginosa: these are termed paAzoR1 (PA0785), binding position would be similar to what was modeled by paAzoR2 (PA1962) and paAzoR3 (PA3223 (Wang et al., Sollner et al. (2009a), where NADH binds in the same pocket 2007)). The three enzymes have significantly different of the active site as balsalazide (Fig. 1A). substrate specificities (Ryan et al., 2010), and we describe here a detailed enzymic characterization of these proteins, Enzyme stability including the NADH quinone oxidoreductase activity of paAzoR3. Although P. aeruginosa lives in relatively mild conditions in soil and on human skin and in the intestinal flora, paAzoR1 RESULTS has previously been shown to be thermostable with a melting temperature (Tm) of approximately 55°C (Wang et al., 2007). Cofactor and hydride donor preference Both paAzoR2 and paAzoR3 show significantly lower melting temperatures when compared to wild type paAzoR1 (40°C Absorbance spectra for each purified enzyme (Fig. 2) show and 50°C, respectively, Fig. 4). Thermostability is not that all three proteins have a typical flavoprotein signature: uncommon among azoreductases, e.g., B. subtilis (Tm absorbance maxima shifted 7–10 nm bathochromically com- 86.5°C) and S. cerevisiae (Tm 60.2°C) determined via circular pared to that of flavin mononucleotide (FMN). The spectra dichroism (Deller et al., 2006). When Tyr131 was mutated to each have a distinct shoulder at λmax of 486 nm as an Phe within the active site of paAzoR1 (equivalent residues are indication of binding of the flavin cofactor to the protein (Fig. 2) prolines in paAzoR2 and paAzoR3), it caused a significant (Duurkens et al., 2007). The flavins are released as indicated shift in Tm to approximately 42°C (Wang et al., 2010a). by disappearance of the shoulder in the spectra upon Oligomerisation state has previously been suggested as a denaturation of the protein and were identified as FMN via possible reason for high thermostability (Deller et al., 2006), TLC (Supplemental Fig. 1). although later work on B. subtilis azoreductase disagreed paAzoR1 is unusual among azoreductases. Unlike many (Binter et al., 2009). Preliminary data from analytical of these enzymes (Nakanishi et al., 2001; Deller et al., 2006), ultracentrifugation experiments on paAzoR2 and paAzoR3 it can utilize both NADH and NADPH as electron donors with (Wang, 2008) indicate they form dimeric and trimeric © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 781 Protein & Cell Ali Ryan et al. Figure 1. Proposed mechanism for balsalazide reduction by paAzoR1. (A) Structure of paAzoR1 binding the prodrug balsalazide (PDB: 3LT5 (Ryan et al., 2010)). Balsalazide is shown in stick formation with a semi-transparent surface, the carbon atoms, which form its azobenzene core, are in white while all other carbon atoms are in orange. The FMN cofactor is in yellow. Important residues lining the active site are shown and labeled.