The Reaction of the Substrate Analog 2-Ketoglutarate with Adenosylcobalamin-dependent Glutamate Mutase

Abstract

Glutamate mutase is one of several adenosylcobalamindependent enzymes that catalyze unusual rearrangements that proceed through a mechanism involving free radical intermediates. The enzyme exhibits remarkable specificity, and so far no molecules other than L-glutamate and L-threo-3-methylaspartate have been found to be substrates. Here we describe the reaction of glutamate mutase with the substrate analog, 2-ketoglutarate. Binding of 2-ketoglutarate (or its hydrate) to the holoenzyme elicits a change in the UV-visible spectrum consistent with the formation of cob(II)alamin on the enzyme. 2-ketoglutarate undergoes rapid exchange of tritium between the 5-position of the coenzyme and C-4 of 2-ketoglutarate, consistent with the formation of a 2-ketoglutaryl radical analogous to that formed with glutamate. Under aerobic conditions this leads to the slow inactivation of the enzyme, presumably through reaction of free radical species with oxygen. Despite the formation of a substrate-like radical, no rearrangement of 2-ketoglutarate to 3-methyloxalacetate could be detected. The results indicate that formation of the C-4 radical of 2-ketoglutarate is a facile process but that it does not undergo further reactions, suggesting that this may be a useful substrate analog with which to investigate the mechanism of coenzyme homolysis.Glutamate mutase catalyzes the reversible isomerization of L-glutamate and L-threo-3-methylaspartate (1-3). It is one of a group of enzymes that use adenosylcobalamin (AdoCbl), 1 a biologically active form of vitamin B 12 , to catalyze unusual 1,2-rearrangements in which an electron-withdrawing group is interchanged with a hydrogen atom on an adjacent carbon (4 -6) The migrating group may be -OH, -NH 2 , or, as in the case of glutamate mutase, a carbon-containing fragment so that a skeletal rearrangement is effected (Fig. 1). The role of AdoCbl as the source of carbon-based radicals, which are unmasked by homolysis of the coenzyme cobalt-carbon bond, is well established (7). Experiments with isotopically labeled substrates and coenzyme have demonstrated that for all the enzymes examined including glutamate mutase, 5Ј-deoxyadenosine acts as the intermediate carrier of the migrating hydrogen (8, 9).Recently, we have shown that in glutamate mutase homolysis of the cobalt-carbon bond and hydrogen abstraction from the substrate are kinetically coupled processes (10). Adenosyl radical can therefore only be formed as a transient high energy species, or may not be formed at all if coenzyme homolysis and hydrogen abstraction are truly concerted processes. Similar findings have been reported for the related AdoCbl-dependent enzyme, methylmalonyl-CoA mutase (11). These unexpected observations have focused our attention on the role of the substrate in initiating free radical formation. A further intriguing and poorly understood aspect of these reactions is how the enzyme controls the rearrangement of substrate-radical to product-radical.In principle, these steps might be probed with substrates that give rise ...

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

“…First, we identified a glutamate mutase reactivatase mutL and expressed this enzyme to regenerate the glutamate mutase from spontaneous inactivation (Zelder et al, 1994b) or inactivation induced by oxygen (Barker et al, 1964), ketoglutarate (Roymoulik et al, 1999) and non-functional form of B12 (CNCbl in our expreiments). Before this report, this has been no previous report on the reactivating factor of glutamate mutase.…”

“…Because of the intrinsic instability and rapid inactivation of the glutamate mutase-AdoCbl complex (Barker et al, 1964;Roymoulik et al, 1999;Toraya, 2003), we suspected that the recombinant expressed glutamate mutase became inactive during fermentation, which led to lower carbon flux to mesaconate. To address this issue, discovery of reactivatase of glutamte mutase would be necessary.…”

“…The characterized proteins include GdrAB, MeaB and PduGH/DdrAB , the reactivatases for glycerol dehydratase (Tobimatsu et al, 1999;Toraya and Mori, 1999), methylmalonyl-CoA mutase (Padovani and Banerjee, 2006) and diol dehydratase (Bobik et al, 1999), respectively. It is reported that glutamate mutase could be inactivated by oxygen (Barker et al, 1964), 2-methyleneglutarate (Huhta et al, 2002) and 2-ketoglutarate (Roymoulik et al, 1999). The spontaneous inactivation of glutamate mutase was observed (Zelder et al, 1994b).…”

Radical Use of Rossmann and TIM Barrel Architectures for Controlling Coenzyme B12Chemistry

“…(S)-2-hydroxyglutarate) (58) or only tritium exchange between substrate and tritiated AdoCbl (e.g. 2-ketoglutarate) (57).…”

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

“…DL-2,4-Diaminobutryic Acid Forms a Stable Radical in OAM-Although many B 12 -isomerases are able to use alternative substrates (24,28), OAM is unable to turn over the competitive inhibitor, DL-2,4-diaminobutryic acid (15). The binding of DAB to holo-OAM probably leads to mechanism-based inactivation of the enzyme that entails AdoCbl-mediated radical propagation, leading to the formation of a PLP-bound radical intermediate.…”