Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N-nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1-2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (k(cat)/K(m)) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine were three orders of magnitude lower. The log k(cat)/K(m) value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [DeltaH(f)(R-O-N(OH)O(-)) or DeltaH(f)(R(1),R(2) > N-N(OH)O(-))], and with an increase in their lipophilicity (octanol/water partition coefficient, log P(ow)), and did not depend on their van der Waals' volumes. Hydrophobic organic nitroesters and hydrophilic N-nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N-nitramines according to their log P(ow) values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes.