Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase.
Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.
Wolterink-van Loo
Suzanne
S
Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.
Siemerink
Marco A J
MA
Perrakis
Georgios
G
Kaper
Thijs
T
Kengen
Servé W M
SW
van der Oost
John
J
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D003080:Cold Temperature; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D008958:Models, Molecular; D015202:Protein Engineering; D016415:Sequence Alignment; D016945:Sulfolobus acidocaldarius / Q000201:enzymology* / Q000378:metabolism
C021470:2-keto-3-deoxygluconate aldolase; D000446:Aldehyde-Lyases