Biotransformation of explosives by the old yellow enzyme family of flavoproteins

Abstract

Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.

FIG. 1.

Transformation of TNT by PETN reductase. Each step requires the oxidation of one equivalent of cofactor.

FIG. 2.

Reactions catalyzed by enzymes from the OYE family. (A) Reductive denitration of PETN. (B) Reductive denitration of GTN. (C) Reduction of TNT (full pathway shown in Fig. 1). (D) Reduction of codeinone. (E) Reduction of cyclohexenone. (F) Reduction of trans-2-hexenal. (G) Reduction of 1-nitrocyclohexene. (H) Reduction of 2-nitrobenzaldehyde.

FIG. 3.

Transformation of TNT by PB2 PETN reductase and OYE. PB2 PETN reductase (A and C) or OYE (B and D) at a concentration of 0.4 μM was incubated with 200 μM TNT, 200 μM NADPH, and a cofactor cycling system. Concentrations of TNT (compound 1, solid circles), HADNTs (compounds 7/8, solid squares), and ADNTs (compounds 9/10, open squares) are shown in panels A and B. Dihydride adduct products (compound 3, open triangles pointing up; compound 4, open triangles pointing down; compound 5, solid triangles pointing up; compound 6, solid triangles pointing down) are shown in panels C and D.

FIG. 4.

Transformation of H⁻-TNT by PB2 PETN reductase and OYE. PB2 PETN reductase (A) or OYE (B) at a concentration of 0.4 μM was incubated with approximately 200 μM H⁻-TNT (compound 2, open circles), 200 μM NADPH and a cofactor cycling system. Dihydride adduct products (compound 3, open triangles pointing up; compound 4, open triangles pointing down; compound 5, solid triangles pointing up; compound 6, solid triangles pointing down) were monitored. No products of nitro group reduction (compounds 7 to 10) were observed.

FIG. 5.

Transformation of TNT by the H184N mutant of PB2 PETN reductase. The H184N mutant of PB2 PETN reductase, at a concentration of 0.4 μM (A) or 4 μM (B), was incubated with 200 μM TNT, 200 μM NADPH, and a cofactor cycling system. Concentrations of TNT (compound 1, solid circles), HADNTs (compounds 7/8, solid squares), and ADNTs (compounds 9/10, open squares) were monitored. Only traces of hydride adduct products (compounds 3 to 6) were observed (not shown).

FIG. 6.

Transformation of H⁻-TNT by the H184N mutant of PB2 PETN reductase. The H184N mutant of PB2 PETN reductase at a concentration of 0.4 μM was incubated with approximately 200 μM H⁻-TNT (compound 2, open circles), 200 μM NADPH, and a cofactor cycling system. Dihydride adduct products (3, open triangles pointing up; 4, open triangles pointing down; 5, solid triangles pointing up; 6, solid triangles pointing down) were monitored. No products of nitro group reduction (7 to 10) were observed.