Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae

Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-re ductone intermediate, CH3SCH2CH2-COCH(OH)=CH(OH) (I), in the methionine sal vage pathway. The two enzymes are remarkable in that they have the same pol ypeptide sequence but bind different metal ions (Ni2+ and Fe2+, respectivel y). ARD converts I to CH3SCH2CH2COOH, CO, and HCOOH. ARD' converts I to CH3 SCH2CH2COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' ha ve ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda (max) red shift upon binding. The dianion formation from II causes the same lambda (max) r ed shift, suggesting that II bind to the enzyme as a dianion. The electron- rich II dianion likely reacts with O-2 to form a peroxide anion intermediat e. Previous O-18(2) and C-14 tracer experiments established that ARD incorp orates O-18(2) into C-1 and C-3 Of II and C-2 is released as CO. ARD' incor porates O-18(2) into C-1 and C-2 Of II. The product distribution seems to n ecessitate the formation of a five-membered cyclic peroxide intermediate fo r ARD and a four-membered cyclic peroxide intermediate for ARD'. A model ch emical reaction demonstrates the chemical and kinetic competency of the pro posed five-membered cyclic peroxide intermediate. The breakdown of the four -membered and five-membered cyclic peroxide intermediates gives the ARD' an d ARD products, respectively. The nature of the metal ion appears to dictat e the attack site of the peroxide anion and, consequently, the different cy clic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turn overs, providing evidence that a radical mechanism may be involved in the f ormation of the peroxide anion intermediate.