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.

Exeter | ERASE

Team:Exeter/DegradationConstructs

From 2014.igem.org

The Solution: Old Yellow Enzymes