Evolution of Enzymatic Activities in the Enolase Superfamily:Galactarate Dehydratase III from Agrobacterium tumefaciens C58

摘要

The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry46, 9564–9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry48, 11546–11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the protonα to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to thebenzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarateas a carbon source under the conditions tested in this study, althoughit does utilize d-galacturonate, which is a likely precursorto m-galactarate. The gene encoding A9CG74 and severalgenome proximal genes were upregulated with d-galacturonateas the carbon source. One of these, a member of the dihydrodipicolinatesynthase superfamily, catalyzes the dehydration and subsequent decarboxylationof 2-keto-3-deoxy-d-galactarate to α-ketoglutaratesemialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.