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. 2007 May 1;403(3):421-30.
doi: 10.1042/BJ20061419.

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

Affiliations

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

Suzanne Wolterink-van Loo et al. Biochem J. .

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.

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Figures

Figure 1
Figure 1. Protein sequence alignment of different members of the NAL subfamily
Alignment of the three Sulfolobus KDGAs with Thermoproteus tenax KD(P)G aldolase, hypothetical KDGAs from Thermoplasma acidophilum (Tac-KDGA), Thermoplasma volcanium (Tvo-KDGA) and Picrophilus torridus (Pto-KDGA), NALs from E. coli (Eco-NAL) and H. influenzae (Hin-NAL), and the E. coli DHDPS (Eco-DHDPS). Residues that are conserved between the KD(P)G aldolases are indicated with dark-grey shading, while those residues that are also conserved in the hypotheticals and other aldolases are shaded in a lighter grey. The arrow indicates the conserved catalytic lysine residue. Catalytic residues (▼) and residues forming the putative phosphate-binding motif in Sac-KDGA (♦) are indicated.
Figure 2
Figure 2. Overall structure of Sac-KDGA and reaction mechanism
(A) Overall structure of Sac-KDGA tetramer. (B) Orthogonal views of the Sac-KDGA monomer. The Schiff-base-forming Lys-153 is indicated in stick presentation with pyruvate (yellow) attached. To show the layout of the substrate-binding pocket, the loop of the neighbouring subunit carrying Arg-105 is also shown, together with Arg-105 and Arg-234 (orange). (C) General scheme of the aldol condensation reaction catalysed by the KDGAs.
Figure 3
Figure 3. Stereo views of the Sac-KDGA active site
(A) Weighted difference density map of pyruvate (green) bound to Lys-153 in the pyruvate-binding cavity of Sac-KDGA. Map is contoured at 4 σ. (B) The substrate-binding pocket of Sac-KDGA with pyruvate. Strands β7 and β8 are indicated. (C) Superposition of the Pyr–Sac-KDGA (green/grey) and KDG–Sso-KDGA complex ([25], yellow/cyan). Hydrogen-bonding interactions with the pyruvate moiety have been omitted for clarity, and the orientation is slightly different from that in (B). Water molecules shown are from the KDG–Sso-KDGA complex. (D) KDPG (yellow) modelled in the active site of Sac-KDGA on the basis of the KDG–Sso-KDGA complex [25]. Water molecules from the crystal structure of native Sac-KDGA, which would have favourable interactions with the ligand are also shown. Some hydrogen-bonding interactions have been omitted for clarity and the orientation is slightly different from that of (B) and (C).

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References

    1. Machajewski T. D., Wong C. H. The catalytic asymmetric aldol reaction. Angew. Chem. Int. Ed. Engl. 2000;39:1352–1375. - PubMed
    1. Samland A. K., Sprenger G. A. Microbial aldolases as C-C bonding enzymes: unknown treasures and new developments. Appl. Microbiol. Biotechnol. 2006;71:253–264. - PubMed
    1. Gijsen H. J., Qiao L., Fitz W., Wong C. H. Recent advances in the chemoenzymatic synthesis of carbohydrates and carbohydrate mimetics. Chem. Rev. 1996;96:443–474. - PubMed
    1. Takayama S., McGarvey G. J., Wong C. H. Microbial aldolases and transketolases: new biocatalytic approaches to simple and complex sugars. Annu. Rev. Microbiol. 1997;51:285–310. - PubMed
    1. Brock T. D., Brock K. M., Belly R. T., Weiss R. L. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mikrobiol. 1972;84:54–68. - PubMed

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