Structure and substrate specificity determinants of NfnB, a dinitroaniline herbicide-catabolizing nitroreductase from Sphingopyxis sp. strain HMH.

Nitroreductases are emerging as attractive bioremediation enzymes, with substrate promiscuity toward both natural and synthetic compounds. Recently, the nitroreductase NfnB from Sphingopyxis sp. strain HMH exhibited metabolic activity for dinitroaniline herbicides including butralin and pendimethalin, triggering the initial steps of their degradation and detoxification. However, the determinants of the specificity of NfnB for these herbicides are unknown. In this study, we performed structural and biochemical analyses of NfnB to decipher its substrate specificity. The homodimer NfnB is a member of the PnbA subgroup of the nitroreductase family. Each monomer displays a central α + ß fold for the core domain, with a protruding middle region and an extended C-terminal region. The protruding middle region of Val75-Tyr129 represents a structural extension that is a common feature to members of the PnbA subgroup and functions as an opening wall connecting the coenzyme FMN-binding site to the surface, therefore serving as a substrate binding site. We performed mutational, kinetic, and structural analyses of mutant enzymes and found that Tyr88 in the middle region plays a pivotal role in substrate specificity by determining the dimensions of the wall opening. The mutation of Tyr88 to phenylalanine or alanine caused significant changes in substrate selectivity toward bulkier dinitroaniline herbicides such as oryzalin and isopropalin without compromising its activity. These results provide a framework to modify the substrate specificity of nitroreductase in the PnbA subgroup, which has been a challenging issue for its biotechnological and bioremediation applications.

Anaerobic reduction of 2,6-dinitrotoluene by Shewanella oneidensis MR-1: Roles of Mtr respiratory pathway and NfnB.

Dinitrotoluene (DNT) is a widely present pollutant in aquatic environments, and its biodegradation is an economically attractive way to effectively removal. In aquatic environments, the presence of electrochemically active bacteria (EAB) could contribute to the anaerobic bioreduction of DNT. However, the mechanism behind such a biodegradation process at gene level remains to be further elucidated. In this work, the anaerobic reduction of 2,6-dinitrotoluene (2,6-DNT) by Shewanella oneidensis MR-1, a typical EAB in aquatic environments, was investigated. S. oneidensis MR-1 was found to be able to obtain energy for growth through the anaerobic respiration on 2,6-DNT. Experimental results show that the Mtr respiratory pathway, a transmembrane electron transport chain, was involved in the 2,6-DNT bioreduction. Knockout of cymA or nfnB resulted in a substantial loss of its 2,6-DNT-reducing ability, indicating that both CymA and NfnB were the key proteins in the microbial electron transfer chain. The genetic analysis further confirms that the Mtr respiratory pathway and NfnB are mainly responsible for the anaerobic reduction of 2,6-DNT by S. oneidensis MR-1. This work is useful to better understand the anaerobic bioreduction of nitroaromatic compounds in aquatic environments and remediate the environments contaminated by nitroaromatic compounds. Biotechnol. Bioeng. 2017;114: 761-768. © 2016 Wiley Periodicals, Inc.

Deep population structure linked to host vernalization requirement in the barley net blotch fungal pathogen.

Invasive fungal pathogens pose a substantial threat to widely cultivated crop species, owing to their capacity to adapt to new hosts and new environmental conditions. Gaining insights into the demographic history of these pathogens and unravelling the mechanisms driving coevolutionary processes are crucial for developing durably effective disease management programmes. Pyrenophora teres is a significant fungal pathogen of barley, consisting of two lineages, Ptt and Ptm, with global distributions and demographic histories reflecting barley domestication and spread. However, the factors influencing the population structure of P. teres remain poorly understood, despite the varietal and environmental heterogeneity of barley agrosystems. Here, we report on the population genomic structure of P. teres in France and globally. We used genotyping-by-sequencing to show that Ptt and Ptm can coexist in the same area in France, with Ptt predominating. Furthermore, we showed that differences in the vernalization requirement of barley varieties were associated with population differentiation within Ptt in France and at a global scale, with one population cluster found on spring barley and another population cluster found on winter barley. Our results demonstrate how cultivation conditions, possibly associated with genetic differences between host populations, can be associated with the maintenance of divergent invasive pathogen populations coexisting over large geographic areas. This study not only advances our understanding of the coevolutionary dynamics of the Pt-barley pathosystem but also prompts further research on the relative contributions of adaptation to the host versus adaptation to abiotic conditions in shaping Ptt populations.

The Pseudomonas putida NfnB nitroreductase confers resistance to roxarsone.

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox) has been used for decades as an antimicrobial growth promoter for poultry and swine. Roxarsone is excreted in chicken manure unchanged and can be microbially transformed into a variety of arsenic-containing compounds such as 3-amino-4-hydroxyphenylarsonic acid (HAPA(V)) that contaminate the environment and present a potential health hazard. To cope with arsenic toxicity, nearly every prokaryote has an ars (arsenic resistance) operon, some of which confer resistance to roxarsone. Pseudomonas putida KT2440 is a robust environmental isolate capable of metabolizing many aromatic compounds and is used as a model organism for biodegradation of aromatic compounds. Here we report that P. putida KT2440 (ΔΔars) in which the two ars operons had been deleted retains resistance to highly toxic trivalent Rox(III), the likely active form of roxarsone. In this study, a genomic library constructed from P. putida KT2440 (ΔΔars) was used to screen for resistance to Rox(III) in Escherichia coli. One gene, termed, PpnfnB, was identified that encodes a putative 6,7-dihydropteridine reductase. Cells expressing PpnfnB reduce the nitro group of Rox(III), and purified NfnB catalyzes FMN-NADPH-dependent nitroreduction of Rox(III) to less toxic HAPA(III). This identifies a key step in the breakdown of synthetic aromatic arsenicals.