PMID:24926996

Citation	Groninger-Poe, FP, Bouvier, JT, Vetting, MW, Kalyanaraman, C, Kumar, R, Almo, SC, Jacobson, MP and Gerlt, JA (2014) Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58. Biochemistry 53:4192-203
Abstract	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) Biochemistry 46, 9564-9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry 48, 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 proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.
Links	PubMed PMC4081050 Online version:10.1021/bi5005377
Keywords	Agrobacterium tumefaciens/enzymology; Agrobacterium tumefaciens/genetics; Amino Acid Sequence; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Crystallography, X-Ray; Genome, Bacterial; Hexuronic Acids/metabolism; Hydro-Lyases/chemistry; Hydro-Lyases/genetics; Hydro-Lyases/metabolism; Molecular Docking Simulation; Molecular Sequence Data; Mutation; Stereoisomerism; Sugar Acids/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHIE6:B3Q5L5		GO:0008867: galactarate dehydratase activity	ECO:0000314:		F		Table 2	complete CACAO 10341
RHIE6:B3Q5L5	enables	GO:0008867: galactarate dehydratase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
AGRRK:B9JNP7		GO:0008867: galactarate dehydratase activity	ECO:0000314:		F		Table 2	complete CACAO 10340
AGRRK:B9JNP7	enables	GO:0008867: galactarate dehydratase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
AGRFC:GCI	Contributes to	GO:0003824: catalytic activity	ECO:0000269:		F		A9CG74 has a DxH motif at the end of β-strand 3 similar to the catalytic residues in D-galactonate dehydratases (Figure 1) but does not catalyze the dehydration of D-galactonate; D-galactonate dehydratases do not catalyze the dehydration of m-galactarate or D-galacturonate. An alignment of the sequences in this cluster shows that both the active site catalytic residues and substrate binding residues in the capping domain are conserved (Figure 1). The enzymes in this cluster likely are orthologues based on sequence identity and shared catalytic/ substrate specificity-determining residues.	complete
AGRFC:A9CG74		GO:0008867: galactarate dehydratase activity	ECO:0000314:		F		Table 2	complete CACAO 10339
AGRFC:A9CG74	enables	GO:0008867: galactarate dehydratase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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