Role of tyrosine 131 in the active site of paAzoR1, an azoreductase with specificity for the inflammatory bowel disease prodrug balsalazide

Abstract

Azoreductase 1 from Pseudomonas aeruginosa strain PAO1 (paAzoR1) catalyses the activation of the prodrug balsalazide and reduces the azo dye methyl red using reduced nicotinamide adenine dinucleotide cofactor as an electron donor. To investigate the mechanism of the enzyme, a Y131F mutation was introduced and the enzymic properties of the mutant were compared with those of the wild-type enzyme. The crystallographic structure of the mutant with methyl red bound was solved at 2.1 A resolution and compared with the wild-type structure. Tyr131 is important in the architecture of the active site but is not essential for enzymic activity.

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…The K m value for DCIP with paAzoR1 is similar to those obtained for azo compounds: 85.3 ± 3.6 μM for DCIP compared to 98.6 μM for balsalazide (Wang et al, 2010a). The V max value is similar to those observed for azo substrates: V max = 0.53 ± 0.01 μM/s for DCIP compared to 0.81 μM/s for balsalazide (Wang et al, 2010a).…”

“…The azoreductase reaction is proposed to be catalyzed via a Bi-Bi ping-pong mechanism (Nakanishi et al, 2001;Liu et al, 2008;Wang et al, 2010a), and we have indicated that the reaction is likely to occur via reduction of the hydrazone tautomer of the substrate, as identified from structural studies ( Fig. 1A) (Ryan et al, 2010).…”

“…Unlike many of these enzymes (Nakanishi et al, 2001;Deller et al, 2006), it can utilize both NADH and NADPH as electron donors with a preference for NADPH (Wang et al, 2007). Interestingly, this preference was reversed on mutation of Tyr131 to phenylalanine in the enzyme's active site (Wang et al, 2010a). In contrast to paAzoR1, which shows a preference for NADPH as the hydride donor, both paAzoR2 and paAzoR3 show a preference for NADH (Fig.…”

“…Thermostability is not uncommon among azoreductases, e.g., B. subtilis (T m 86.5°C) and S. cerevisiae (T m 60.2°C) determined via circular dichroism (Deller et al, 2006). 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).…”

Bacterial diversity and composition in major fresh produce growing soils affected by physiochemical properties and geographic locations

“…Two broad types of azoreductases including flavin-dependent and non-flavin reductases have been reported (Blumel & Stolz, 2003;Maier et al, 2004;Suzuki et al, 2001). Flavin-dependent azoreductases usually reduce azo dyes through a Ping-Pong Bi-Bi mechanism (Nakanishi et al, 2001;Ryan et al, 2010;Wang et al, 2010). In this mechanism, two cycles of NADPH-dependent reduction of FMN to FMNH occur, converting azo dye to a hydrazine in the first step, and then the hydrazine to two constitutive amines in the second step (Correia et al, 2011;Ryan et al, 2010).…”