Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases

Abstract

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…This was found to be correlated with the presence of a newly identified phosphate binding motif comprised of two conserved Arg residues from two neighboring subunits of the tetramer. Such a phosphate binding motif was interestingly not found in nonphosphorylating KDGA (not accepting KDPG or GAP as the substrate) (297). In addition to the C 3 substrates GA and GAP, the Sulfolobus enzyme converts C 2 substrates like glycolaldehyde and glyoxylate as well as C 4 aldotetroses like D-and L-erythrose and threose.…”

“…acidocaldarius, and Tpt. tenax have been reported (295)(296)(297)(298). They belong to the protein family of Schiff base-forming class I aldolases, in which the archaeal KD(P)G aldolases appear to belong to a distinct subgroup of enzymes (SCOP database).…”

Cohesion Group Approach for Evolutionary Analysis of TyrA, a Protein Family with Wide-Ranging Substrate Specificities

“…These include (i) a detailed assessment of the basis for the catalytic promiscuity of E. coli alkaline phosphatase, which can also act as a sulfatase (16); (ii) a new family of lactonases that hydrolyze a variety of lactones, possess low phosphotriesterase activities, and have been shown to be the source of a newly evolved and highly efficient phosphotriesterase (2); (iii) a gentisate dioxygenase that also functions with 1,4-dihydroxy-2-napthoate and salicylate (31); (iv) an ATP-dependent hexokinase from Sulfolobus tokadaii that can phosphorylate glucose, mannose, glucosamine, and N-acetylglucosamine (54); (v) a higher-plant isopropylmalate synthase that not only condenses acetyl coenzyme A (acetyl-CoA) with 2-ketoisovalerate but will also accept 2-oxo acid substrates of two-carbon to six-carbon lengths (19); (vi) a number of variations in the substrate specificities of glutathione synthesis enzymes in comparison to E. coli, Streptococcus agalactiae, and Clostridium acetobutylicum (42); (vii) an amino acid racemase from Pseudomonas putida with an unusual breadth of specificity for amino acids (43); (viii) ATP-forming acetyl-CoA synthetases that accept acetate, propionate, and some longer straight-and branched-chain acyl substrates (32); (ix) an isochorismate pyruvate lyase from Pseudomonas aeruginosa that also has weak chorismate mutase activity (45); and (x) Sulfolobus species that condense pyruvate and aldehydes with two to four carbon atoms (phosphorylated or not) (74). D-2-Hydroxyacid dehydrogenase from Haloferax mediterranei exhibits interesting parallels to the broad-specificity TyrA variants.…”

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

“…A 16 lL sample was used to determine the remaining pyruvate as described above. Reactions with the aldoses as substrates were additionally analyzed with a thiobarbituric acid-assay as described previously (Wolterink-van Loo et al 2007). …”