Misal and Gawai Bioresour. Bioprocess. (2018) 5:17 https://doi.org/10.1186/s40643-018-0206-8 REVIEW Open Access Azoreductase: a key player of xenobiotic metabolism Santosh A. Misal1,2* and Kachru R. Gawai1* Abstract Azoreductases are diverse favoenzymes widely present among microorganisms and higher eukaryotes. They are mainly involved in the biotransformation and detoxifcation of azo dyes, nitro-aromatic, and azoic drugs. Reduction of azo bond and reductive activation of pro-drugs at initial level is a crucial stage in degradation and detoxifca- tion mechanisms. Using azoreductase-based microbial enzyme systems that are biologically accepted and ecof- riendly demonstrated complete degradation of azo dyes. Azoreductases are favin-containing or favin-free group of enzymes, utilizing the nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a reducing equivalent. Azoreductases from anaerobic microorganisms are highly oxygen sensitive, while azoreduc- tases from aerobic microorganisms are usually oxygen insensitive. They have variable pH, temperature stability, and wide substrate specifcity. Azo dyes, nitro-aromatic compounds, and quinones are the known substrates of azore- ductase. The present review gives an overview of recent developments in the known azoreductase enzymes from diferent microorganisms, its novel classifcation scheme, signifcant characteristics, and their plausible degradation mechanisms. Keywords: Azo dye, Azoreductase, Bioremediation, Biotransformation, Detoxifcation, Xenobiotics Introduction of physical, chemical, and biological treatment proce- Azo dyes and nitro-aromatic compounds are consid- dures are employed to degrade and detoxify the chemi- ered as potential xenobiotics. Tey are extensively used cal content and to remove color from dye-containing worldwide in textile, paint, printing, cosmetics, and phar- industrial wastewater. Te complex aromatic structures maceutical industries. A high discharge of untreated of azo dyes and nitro-aromatic compounds are not being wastewater from these industries is the major source of efciently degraded by conventional treatment methods. azo dyes to enter into the ecosystem (McMullan et al. Biological treatment methods include microbial bio- 2001; Stolz 2001). Azo dyes containing nitro and amine degradation in aerobic, anaerobic, anoxic, or combined moieties are toxic and mutagenic to biological systems. anaerobic/aerobic conditions (McMullan et al. 2001; Synthetic nitro-aromatic compounds are also potential Robinson et al. 2001). However, all of these methods have mutagenic and carcinogenic to biological system (Chung limitations and some drawbacks. It has been proven that and Cerniglia 1992; Rafi and Cerniglia 1995). the use of one individual process may often be not suf- A typical azo dye contains one or more characteristic fcient to achieve complete decolorization or minerali- azo bonds (–N=N–) in their complex aromatic structure. zation of dye. Terefore, the dye degradation strategies Tis semicovalent azo linkage makes azo dyes more recal- should consist of a combination of diferent physical, citrant to microbial degradation, and it is not readily bro- chemical, as well as biological techniques. Recently, some ken down under the environmental conditions. Varieties investigators demonstrated that the biological dye degra- dation techniques by pure and mixed cultures of bacte- *Correspondence: [email protected]; [email protected] ria, fungi, and algae are more useful, and technically and 1 Biochemistry Division, Department of Chemistry, Savitribai Phule Pune economically feasible. Microbial enzyme-based technol- University, Pune 411 007, India ogies would also be highly efcient methods for remov- 2 Present Address: Department of Chemistry, Indiana University, Bloomington, IN 47405, USA ing xenobiotics from environment (Stolz 2001; Robinson © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Misal and Gawai Bioresour. Bioprocess. (2018) 5:17 Page 2 of 9 et al. 2001; Bürger and Stolz 2010; Oturkar et al. 2013). (Wang et al. 2010; Ryan et al. 2010a). In all the suggested Utilization of diferent microbial oxidases and reductases pathways, azoreductases are involved in the reductive demonstrated the complete dye degradation at experi- cleavage of the azo bond as the initial stage in degrada- mental conditions (Lang et al. 2013; Oturkar et al. 2013). tion of azo dyes. Tis has been the topic of interest in the In higher organisms, degradation of azo compounds recent years as these enzymes are also involved in carci- is considered to be a detoxifcation metabolic pathway. nogenic compound metabolism and their cleanup from Most of the azo compounds are considered as potent environment. Indeed, in all these processes, the precise pre-carcinogens. Te metabolites generated after the biochemical role and the mechanism of azoreductase are breakdowns of azo bonds are supposed to be non-carci- still not well explored. nogenic, but in some cases, it may lead to be more car- Tis review presents an overview and evaluation of the cinogenic (Stolz 2001). Once the azo compounds enter properties of the known azoreductase enzymes from dif- into the human body through ingestion, inhalation, or ferent microorganisms, and sheds a light on its signif- skin contact, they are initially metabolized via azoreduc- cant characteristics. Moreover, it can also provide new tases to aromatic amines in the skin and the gastroin- insights into its new classifcation scheme and demon- testinal tract, and further metabolized through the liver strate the versatile nature in biodegradation, biotransfor- (Rafi and Cerniglia 1995). Te human gastrointestinal mation, and detoxifcation of xenobiotic procedures. tract contains a complex micro fora comprising at least 400–500 bacterial species. Some of them are isolated and Azoreductase classifcation characterized with high activity of azo nitroreductase in Azoreductases are the diverse group of enzymes widely the presence of favins and NAD(P)H (Rafi and Cerniglia present in a variety of microorganisms with variations in 1995; Koppel et al. 2017). However, the precise role of their structures and functions. Although there is diversity azoreductases in drug metabolism is not yet known. It in structure and function, they have a common potential is well known that azoic drugs are used in the treatment for reduction of azo dyes (Fig. 1). Several azoreductases of infammatory bowel disease. Tey are activated in the have been isolated, purifed, and biochemically char- gut by NAD(P)H quinone oxidoreductase. NAD(P)H qui- acterized from aerobic and anaerobic microorganisms, none oxidoreductase is the human ortholog of azoreduc- and their encoding genes were identifed (Zimmermann tase (Ryan et al. 2010a, b). Moreover, the colon-specifc et al. 1982; Bryant and DeLuca 1991; Punj and John 2009; drug delivery could also be possible by the azoreductase Matsumoto et al. 2010; Bürger and Stolz 2010; Misal sensitive system (Rao and Khan 2013). However, the pre- et al. 2011; Morrison et al. 2012). Azoreductases from cise biochemical mechanism is still unclear. Recently, diferent sources are diverse in their catalytic activity, Ryan et al. (2011) studied the mechanism of azoreduction cofactor requirement, and biophysical characteristics. and the ability of azoreductase to reduce nitro-aromatic Terefore, the interests have been evinced in categorizing and quinone compounds. Te nitro-aromatic com- the known enzymes showing azoreductase activity and pounds are commonly used in nimesulide, nitrofurazone, discovering their common evolutionary origin. and tolcapone, and it may lead to hepatotoxicity upon Abraham and John (2007) initially classifed azoreduc- reduction of a nitro group or azo bond by azoreductase tases on the basis of secondary and tertiary structures of or nitroreductase (Rafi and Cerniglia 1995; Ryan et al. azoreductase predicted from their amino acid sequences. 2010a). Terefore, it is very important to explore the drug Due to the low level of sequence homology, azoreduc- metabolism pathways in detail. Te bacterial azoreduc- tases were further classifed on the basis of oxygen toler- tases are known to be involved in drug metabolism via ance or cofactor requirement, predominantly their favin favin or nicotinamide cofactors. Few metabolic pathways and NADH-dependence (Bafana and Chakrabarti 2008). have been suggested for azo dye degradation and detoxi- Te diversity in gene sequence and structures of aero- fcation in aerobic and anaerobic conditions. An aerobic bic and anaerobic azoreductase appeared to be two dis- pathway involves the azoreductase as the major player tinct azoreductases (Rafi and Coleman 1999; Suzuki and an anaerobic pathway in which the azo compound et al. 2001; Chen et al. 2004). Based on the oxygen toler- reduction is mediated by reduced quinone compounds ance, azoreductases were classifed in two major groups, resulting from quinone reductase activities (Kudlich oxygen-sensitive and oxygen-insensitive azoreductases et al. 1997; Liu et al. 2008; Gonçalves et al. 2013). Liu (Chen et al. 2005; Nakanishi et al. 2001). Bürger and et al. (2009) suggested that the azoreductases