Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

Abstract

The flavin hydroperoxide at the active site of the mixed-function oxidase 2-aminobenzoyl-CoA monooxygenase͞reductase (Azoarcus evansii) transfers an oxygen to the 5-position of the 2-aminobenzoyl-CoA substrate to provide the alkoxide intermediate II ؊ . Hydrogen migration from C5 to C6 follows this monooxygenation. The nature of the monooxygenation intermediate and plausible competing reactions leading to hydrogen migration have been considered. Ab initio molecular orbital theory has been used to calculate structures and electron distributions in intermediate and transition state structures. Electrostatic potential surface calculations establish that the transition state and product, associated with the C5 to C6 hydrogen transfer, are stabilized by electron distribution to the benzoyl-CoA thioester carbonyl oxygen. This is not so for the transition state and product associated with hydrogen transfer from C5 to C4. The activation energy for the 5,6-shift is 2.5 kcal͞mol lower than that for the 5,4-shift. In addition, the product of the hydrogen 5,6-shift is more stable than is the product of the hydrogen 5,4-shift, by Ϸ6 kcal͞mol. These results explain why only the shift of hydrogen from C5 to C6 is observed experimentally. Oxygen transfer and hydrogen migration almost coincide in the gas phase (activation energy of Ϸ0.6 kcal͞mol, equivalent to a single bond vibration). Enzymatic formation of alkoxide II ؊ requires its stabilization; thus, the rate constant for its breakdown would be slower than in the gas phase. O xygenase enzymes are separated into two classes: dioxygenases and mixed-function oxidases. Dioxygenases transfer both oxygens from molecular oxygen to the substrate to provide product. Mixed-function oxidases transfer one oxygen from molecular oxygen to the substrate, and the remaining oxygen is incorporated into a water molecule. For flavin mixed-function oxidases, Hamilton proposed that oxygen reacts with enzymebound dihydroflavin to provide a 4a-hydroperoxyflavin (4a-FlH-OOH) (1). Kemal and Bruice synthesized a 4a-hydroperoxide [4a-hydroperoxy-5-ethyl-3-methyllumiflavin (4a-FlEt-OOH)] by reaction of hydrogen peroxide with the flavinium cation, N(5)-ethyl-3-methyl-1,5-dihydrolumiflavin (2). These studies made it possible to explore oxygen transfer from 4a-hydroperoxides to substrates (3). The reaction of 1,5-dihydroflavins to form 4a-hydroperoxideds has been shown to involve O 2 . and a flavin radical as intermediates (4, 5). It is generally accepted that flavin-4a-hydroperoxides form in the active sites of flavindependent mixed-function oxidases when reduced flavin reacts with molecular oxygen (6-12). It was determined that the monooxygen transfer involving 4a-hydroperoxyflavins does not occur from a carbonyl oxide formed on ring opening of the isoalloxazine ring between C4 and C4a (13, 14), as originally proposed (1), but by nucleophilic attack on the terminal oxygen of the flavin 4a-hydroperoxide (15). Dioxygen transfer from the oxygen anion 4a-FlEt-OO Ϫ is known in model reactions (3,(16)(17)(1...

Steroids, triterpenoids and molecular oxygen

“…Epoxidation proceeds by the reaction of oxygen with the bound dihydroflavin (Fl red H 2 ) to give a 4a-hydroperoxyflavin (FlH(4a)-OOH). The oxygen transfer occurs by nucleophilic attack by the 2,3 double bond of squalene on the terminal oxygen of the 4a-hydroperoxide, yielding oxidized flavin (Fl ox ) and 2,3-oxidosqualene (Bruice et al 1983;Torres & Bruice 1999). The FAD is regenerated by NADPH-cytochrome P450 reductase (Laden et al 2000).…”

Aerobic Benzoyl-Coenzyme A (CoA) Catabolic Pathway in Azoarcus evansii : Conversion of Ring Cleavage Product by 3,4-Dehydroadipyl-CoA Semialdehyde Dehydrogenase

“…This homodimeric 122-kDa enzyme does not require oxygen and catalyzes the following reaction: 2,3-dihydro-2,3-dihydroxybenzoyl-CoA ϩ H 2 O 3 3,4-dehydroadipyl-CoA semialdehyde ϩ HCOOH. It is noteworthy that not only benzoate but also phenylacetate (11,13,25,27) and 2-aminobenzoate (4,18,33,35) were shown in various bacteria to be metabolized via unprecedented CoA-dependent new pathways.…”

Two Similar Gene Clusters Coding for Enzymes of a New Type of Aerobic 2-Aminobenzoate (Anthranilate) Metabolism in the Bacterium Azoarcus evansii

“…The reaction requires 2 NADH molecules and 1 O 2 molecule. ACMR and the reaction it catalyzes have been studied experimentally and theoretically in some detail (12,13,17,22,23,24,30). In addition, its Nterminal amino acid sequence has been determined (2).…”