Difference between revisions of "Part:BBa K216006"

Latest revision as of 16:21, 20 October 2009

pentaerythritol tetranitrate reductase with native rbs

This is the coding sequence for the enzyme pentaerythritol tetranitrate reductase (PETN reductase) of Enterobacter cloacae PB2. This enzyme reductively denitrates nitrate esters such as PETN (pentaerythritol tetranitrate, penthrite) GTN (glycerol trinitrate, nitroglycerine) and EGDN (ethylene glycol dinitrate) yielding the alcohol and nitrite ion. It can also reductively attack nitroaromatic compounds such as TNT (2,4,6-trinitrotoluene) forming initially hydride-Meisenheimer complexes which break down via an unknown route to yield nitrite and non-aromatic products. Reference: Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. (1996). Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Applied and Environmental Microbiology 62(4), 1214-1219. Reference 2: French,C.E., Nicklin,S. and Bruce,N.C. 1996. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2. J. Bacteriol. 178 (22), 6623-6627 (1996).

Usage and Biology

PETN reductase (encoded by the gene onr, for Organic Nitrate Reductase) was isolated from Enterobacter cloacae PB2, an organism isolated from explosive-contaminated soil on the basis of its ability to use pentaerythritol tetranitrate (PETN, penthrite, a common plastic explosive) as sole nitrogen source. PETN reductase is an FMN-binding flavoprotein of the Old Yellow Enzyme family. It is reduced by NADPH, and its main activity is reductive denitration of nitrate esters such as PETN and GTN (glyceryl trinitrate, or nitroglycerine). The nitrogen is liberated as nitrite anion (NO2-). Nitrate esters with 3 or more nitrate groups are denitrated much faster than those with two or fewer; thus PETN is rapidly denitrated to pentaerythritol trinitrate and then pentaerythritol dinitrate, but further denitration is much slower. PETN reductase operates by a ping-pong mechanism. Kinetic parameters were measured as:

• Vmax = 25.2 +/- 1.5 U/mg (one unit produces 1 micromole nitrite per minute under assay conditions)
• kcat = 16.6 +/- 1.0 s-1
• K (NADH) = 107 +/- 10 micromolar
• K (GTN) = 39.3 +/- 3.5 micromolar
• KAR = 400 +/- 58 micromolar (this is the dissociation constant for unproductive binding of NADPH to the reduced form of the enzyme).

Figures are taken from French et al (1996). It was later found that PETN reductase was also active against nitroaromatic explosives such as TNT (2,4,6-trinitrotoluene) (French et al, 1998). Unlike other small flavoproteins, which generally act as nitroreductases (reducing the aromatic nitro groups to nitroso groups, then hydroxylamino groups, then amine groups, a very facile reaction due to the powerful electron-withdrawing nature of the nitro groups), PETN reductase was found to liberate nitrogen as nitrite from TNT, and to produce non-aromatic products. While the parthway for this was not fully elucidated, the initial step appeared to be reduction of TNT by transfer of a hydride from reduced PETN reductase to form a dark red hydride-Meisenheimer complex, followed by a rapid second reduction to generate an orange dihydride complex (there are two possible initial hydride adducts and two possible dihydride adducts). This orange product eventually broke down to undetermined products with elimination of one nitrogen as nitrite. It was later reported that PETN reductase might also have some activity against the third major class of secondary high explosives, the nitramines, such as RDX (Royal Demolition Explosive, cyclonite, hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (High Melting Explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine). Thus PETN reductase appears to be uniquely suited to two applications - biosensors for detection of explosives, based on NADPH oxidation, or, more sensitively, nitrite release; and bioremediation of explosive residues in soil.

Sequence and Features