Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases.
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.
Wolterink-van Loo
Suzanne
S
Laboratory of Microbiology, Wageningen University, 6703 CT Wageningen, The Netherlands. suzanne.vanloo@wur.nl
van Eerde
André
A
Siemerink
Marco A J
MA
Akerboom
Jasper
J
Dijkstra
Bauke W
BW
van der Oost
John
J
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D011773:Pyruvates / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
D000447:Aldehydes; D011773:Pyruvates; D000446:Aldehyde-Lyases; C021468:2-keto-3-deoxy-D-glucarate aldolase