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Contents 1 The Solution: Old Yellow Enzymes 2 XenB 2.1 Degradation of TNT 2.2 Degradation of Nitroglycerin

The Solution: Old Yellow Enzymes

From our wider research, it was clear that there was a serious need, both from an environmental and humanitarian perspective, to develop solutions to rapid identification and long-term degradation of explosives. Thus, we initiated research into the microbial TNT and Nitroglycerin attacking enzymes that have been discovered and whether other biobricks or industrial products had been generated.

Several microbial enzymes have the ability to catalyse the break-down of nitroaromatic compounds such as TNT and NG. These enzymes fall into two main families:

• Oxygen-Insensitive Nitroreductases: These enzymes sequentially perform two-electron reductions of nitro groups. They typically contain Flavin mononucleotides FMN which they use, along with NADPH as a cofactor and electron donor. Examples include the nitroreductases NfsA and NfsB from Escherichia coli1, PnrA and PnrB from Pseudomonas putida2 , and NitA and NitB from Clostridium acetobutylicum3.
• Old Yellow Enzymes (OYE): The physiological function of this family of NADPH dehydrogenases is not yet well established; however, they are often associated with nitroaromatic compound reduction. Within the OYE family, two types of enzymes have been described:
• Type I hydride transferases, which, like the oxygen-insensitive nitroreductases above, reduce the nitroaromatic compounds to hydroxylamine derivatives,
• Type II hydride transferases, which catalyse a nucleophilic attack on the aromatic ring of TNT4,5

Of bacterial OYE family members, those that are the best characterized are XenA - XenF of P. putida KT24406 XenB from P. fluorescens7, PETN reductase from Enterobacter cloacae PB28, NemA reductase from E. coli9 and YqjM from Bacillus subtilis10.

In 2009 the Edinburgh iGEM team developed the concept of a Nitrate/Nitrite biosensor which could be used to detect TNT. They generated a biobrick (BBa_K216006) for the gene onr (organic nitrate reductase) that encodes pentaerythritol tetranitrate (PETN) reductase to function as a TNT degrader. However, neither were properly characterised.

We sought to find new, mostly uncharacterised, enzymes to function as additional solutions to offer alternative mechanisms which may be more suited to particular problems. Given that the PETN reductase was a member of the Old Yellow Enzyme family, we decided that we would target additional members of the OYE group to help expand the both the range of enzymes that can be utilised to degrade explosives and the range of UXO chemicals that could be degraded to those that contain nitroglycerin.

Our initial shortlist contained the following the proteins: XenA, XenB, NemA and YqjM . From these we selected two to examine over the summer. These were XenB and NemA.

XenB

XenB is an NADH-dependent flavoprotein (Xenobiotic Reductase B) from the soil bacterium Pseudomonas fluorescens. We chose XenB because: 1. XenB may serve a dual purpose as a Nitroglycerin and TNT degrading enzyme (see below). 2. The phylogeny of OYE members11 suggests that XenB is similar in sequence to the previously submitted PETN reductase and is comparatively well understood. 3. Heterologous expression of XenB from P. fluorescens has been previously demonstrated in E. coli DH5α12 enhancing our chances of successful expression.

Degradation of TNT

Pak et al. (2000)14 reported the purification of the NADH-dependent flavoprotein oxidoreductase xenobiotic reductase B (XenB) from Pseudomonas fluorescens.

Those authors provide a guide to some of the degradative reactions of XenB from P. fluorescens. This indicates that XenB may catalysed the reduction of TNT, either by adding a hydride to the aromatic ring and forming a dihydride Meisenheimer complex15 or by catalysing the reduction of nitro groups directly (Figure 1). However several unidentified products and proposed intermediates indicate that this process is yet to be fully elucidated.

XenB from Pseudomonas fluorescens is not the only XenB enzyme that exists. A more commonly studied XenB enzyme is found in Pseudomonas putida. This XenB shares 88% identity to our chosen XenB from P. fluorescens16. XenB from P. putida (as well as other xenobiotic reductases) demonstrate type II hydride transferase activity against TNT. Research into these xenobiotic reductases provided a greater understanding of the potential products and intermediates generated in the reactions with TNT (Figure 3). Using figure 1 as a model, in the mechanisms suggested by Pak et al. (2000) (above) it seems likely that the unknown m/z 196 compound was 4ADNT and that the proposed bridge product (m/z= 376) is instead linked by an amine group bridge (Figure 2).

We were unable to find more recent attempts to characterise the mechanism of XenB from P. fluorescens. However, given that these mechanisms for P. putida Xenobiotic reductase proteins are similar to the mechanisms of the type II hydride transferase family18 it suggests we are more likely to find the amine bridged products in Figure 2.

Degradation of Nitroglycerin

XenB also displays an apparent capacity to denitrify Nitroglycerin (NG). It has been demonstrated that XenB catalyses the NADH-dependent cleavage of nitro groups from NG, releasing nitrite (Figure 3). P. fluorescens XenB also exhibits five-fold regioselectivity for removal of the central nitro group from NG compared to P. putida XenB.

As nitrate is released from the degradation of Nitroglycerin there is a suggestion that construct could be built upon to work simultaneously as an explosive degrader and soil enricher, thus taking an environmental toxicant and converting it into an environmental benefit.

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