Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives

Abstract

Chloramphenicol nitroreductase (CNR), a drug-modifying enzyme from Haemophilus influenzae, has been shown to be responsible for the conversion of the nitro group into an amine in the antibiotic chloramphenicol (CAM). Since CAM structurally bears a 4-nitrobenzene moiety, we explored the substrate promiscuity of CNR by investigating its nitroreduction of 4-nitrobenzyl derivatives. We tested twenty compounds containing a nitrobenzene core, two nitropyridines, one compound with a vinylogous nitro group, and two aliphatic nitro compounds. In addition, we also synthesized twenty-eight 4-nitrobenzyl derivatives with ether, ester, and thioether substituents and assessed the relative activity of CNR in their presence. We found several of these compounds to be modified by CNR, with the enzyme activity ranging from 1-150% when compared to CAM. This data provides insights into two areas: (i) chemoenzymatic reduction of select compounds to avoid harsh chemicals and heavy metals routinely used in reductions of nitro groups and (ii) functional groups that would aid CAM in overcoming the activity of this enzyme.

Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens

“…Previous work has demonstrated that the NfsB enzyme from H. influenzae strain Rd KW20 (Hi KW20) has the ability to reduce chloramphenicol to amino-chloramphenicol in vitro ( 15 – 17 ). Smith et al initially identified several Haemophilus strains, including Hi KW20, and taxonomically unrelated bacterial strains as having the ability to reduce chloramphenicol in bacterial culture ( 12 ).…”

“…This satisfies the definitions of cryptic and proto-resistance genes and shows potential for clinical relevance through mobilization/change in context and change in expression ( 23 ). Here, we built upon this initial observation ( 15 – 17 ) to survey the functional landscape of potential chloramphenicol reductases across a range of amino acid identities.…”

“…Recently, we found that a Haemophilus influenzae homolog of the NfsB nitroreductase enzyme can confer clinical levels of antibiotic resistance when expressed in Escherichia coli ( 15 ). We and others have demonstrated that this enzyme, specifically from H. influenzae strain Rd KW20, can reduce the nitro group of chloramphenicol to the corresponding amine in vitro ( 15 – 17 ). It is via this promiscuous nitro reductase activity that NfsB confers chloramphenicol resistance.…”

Genomic analysis of the Bacillus megaterium Mes11: New insights into nitroreductase genes associated with the degradation of mesotrione

“…Hence, our results provide new insight into the mesotrione transformation by Bacillus megaterium Mes11 strain and highlighted the key physiological role of the NfrA2 enzyme in this process. Nitroreductases being involved in reduction of various nitro-aromatic compounds such as pesticides and antibiotics (Caballero et al, 2005;Symons and Bruce, 2006;Roldán et al, 2008;Carles et al, 2016;El-Hossary et al, 2017;Green et al, 2019), these enzymes (or their genes) could be used as biomarkers to predict the capacity of ecosystems to transform such molecules.…”

Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

“…It was first reported in the 1980s that certain bacterial species might be able to evade the toxicity of chloramphenicol through nitro-reduction (Yunis, 1988). More recently it has been proven that a nitroreductase from Haemophilus influenzae is capable of conferring Niclosamide Chloramphenicol chloramphenicol resistance through reduction of the nitro-group (Smith et al, 2007;Crofts et al, 2019;Green, 2019).…”