Mechanistic Studies of Two Dioxygenases in the Methionine Salvage Pathway of Klebsiella pneumoniae^,

Abstract

Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have 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) red 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 intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.

Targeting a Novel Plasmodium falciparum Purine Recycling Pathway with Specific Immucillins

“…A pathway for conversion of MTR-1-P to methionine is expressed in bacteria and mammals, although the pathway is not fully characterized in any organism (16,34) (shown as a dashed arrow in Fig. 6).…”

One Protein, Two Enzymes Revisited: A Structural Entropy Switch Interconverts the Two Isoforms of Acireductone Dioxygenase

“…As such, the activation of dioxygen, an important role for most oxygenases, is likely less important for ARD, and no significant binding of O 2 is observed to the substrate-free enzyme (7). At physiological pH, acireductone is predominantly a monoanion, and acireductone binding to either ARD and ARD′ involves the loss of a second proton, yielding the dianionic species (7).…”

“…The methionine salvage pathway returns the thiomethyl group of S-adenosylmethionine 1 (SAM) via methylthioadenosine 2 (MTA) to a new molecule of methionine in which the other carbon atoms are derived from the ribose ring of 1. The penultimate intermediate in the methionine salvage pathway, 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene 4, contains the acireductone functionality C(O)-C(OH)=CH(OH), and is the substrate for ARD (5)(6)(7). If Fe +2 is bound in the active site (ARD′), the substrate acireductone reacts with O 2 to yield formate and the keto-acid precursor of methionine, 4-methylthio-2-ketobutyrate 5, and formate ( Figure 1).…”

“…ARD and ARD′ are both monomers, and are kinetically stable in that metal replacement is required to switch from one fold to the other. It has been shown (7,8) that Ni +2 in ARD can be conservatively replaced by Mn +2 or Co +2 , giving rise to ARD activity (CO production), while Fe +2 in ARD′ can be replaced (albeit with lower activity) by Mg +2 . This promiscuity towards metal cofactors of widely different reduction potentials suggests that the metal is not an activator of triplet O 2 , but rather acts to control protein structure and ligand orientation so as to direct the reaction between O 2 and acireductone towards a particular regiochemistry (9).…”

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

“…Acireductone dioxygenase (ARD), a metalloenzyme from the methionine salvage pathway of the bacterium Klebsiella ATCC 8724, is unusual in that it catalyzes different dioxygenase reactions with identical substrates depending upon the metal bound in the active site Dai et al, 2001). The methionine salvage pathway returns the γ-thiomethyl group of methylthioadenosine (MTA) to methionine (Kestell, 1996).…”