Part:BBa_K216006

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).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 321
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 783