The crystal structure ofPseudomonas putidaazoreductase - the active site revisited

Abstract

The enzymatic degradation of azo dyes begins with the reduction of the azo bond. In this article, we report the crystal structures of the native azoreductase from Pseudomonas putida MET94 (PpAzoR) (1.60 Å), of PpAzoR in complex with anthraquinone‐2‐sulfonate (1.50 Å), and of PpAzoR in complex with Reactive Black 5 dye (1.90 Å). These structures reveal the residues and subtle changes that accompany substrate binding and release. Such changes highlight the fine control of access to the catalytic site that is required by the ping‐pong mechanism, and in turn the specificity offered by the enzyme towards different substrates. The topology surrounding the active site shows novel features of substrate recognition and binding that help to explain and differentiate the substrate specificity observed among different bacterial azoreductases.

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

“…Flavin contributes thermal stability to azoreductase enzyme (Natalello et al, 2007). In general, flavin enzymes exhibit broad substrate specificity with respect to the chemical structure and size of the dye substrate, making it particularly non-specific for any dye group (Goncalves et al, 2013). The azoreductases are classified into two groups on the basis of their electron donor requirements.…”

“…These changes highlight the fine control and access to the catalytic site that are required by the Ping-Pong mechanism, and in turn, the specificity is offered by the enzyme towards different substrates. The topology surrounding the active site shows novel features of substrate recognition and binding that help to explain and differentiate the substrate specificity observed among different bacterial azoreductases (Goncalves et al, 2013). The monomer and dimer structures of the azoreductases determine their substrate specificity and thermostability (Brissos et al, 2014).…”

Azoreductase: a key player of xenobiotic metabolism

“…Few metabolic pathways have been suggested for azo dye degradation and detoxification in aerobic and anaerobic conditions. An aerobic pathway involves the azoreductase as the major player and an anaerobic pathway in which the azo compound reduction is mediated by reduced quinone compounds resulting from quinone reductase activities (Kudlich et al 1997;Liu et al 2008;Gonçalves et al 2013). Liu et al (2009) suggested that the azoreductases might be involved in the detoxification of quinones.…”

Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes

“…In paAzoR2 and paAzoR3 the negative charge imparted to the FMN after reduction by the NAD(P)H is predicted, based upon homology models [15], to be stabilised by interactions with His144 in the case of paAzoR2 and Tyr145 in the case of paAzoR3. In the case of paAzoR2 the interaction with His144 is supported by the recently published native structure of its homologue (ppAzoR) from P. putida MET94 (71% identical PDB: 4C0W [38]). In contrast in paAzoR1 there is no such stabilisation as the equivalent residue is Phe150 which has no polar group to interact with FMN.…”

“…One contributing factor to the substrate specificity may be structural, paAzoR2 and paAzoR3 are thought to have significantly larger active sites than paAzoR1. The increased size of the active site of paAzoR2 is supported by the native crystal structure of its homologue ppAzoR [38] where the helix bearing Phe60 shifts significantly outwards, away from the FMN, thus increasing the volume of the active site. In the case of paAzoR3, there are no close homologous structures, however a homology model [15] predicts a F60A substitution that would significantly increase the size of the active site.…”