Molecular cloning, sequency, and expression of the heat-labile uracil-DNA glycosylase from a marine psychrophilic bacterium, strain BMTU3346.
The gene encoding a heat-labile uracil-DNA glycosylase (UDG) from a psychrophilic, gram-positive marine strain (BMTU3346) has been cloned, sequenced, and expressed in Escherichia coli. The UDG is a cold-active enzyme with an apparent temperature optimum of 35 degrees C and a half-life of 2min at 40 degrees C. The amino acid sequence shows an identity of 39.1%-46.2% to UDGs from mesophilic bacteria. The primary structure was examined for features that could be related to the thermolability of the enzyme. The amino acid sequence of the heat-labile UDG shows 22 differences with respect to the consensus sequence derived from bacterial UDGs. Features previously recognized in cold-active enzymes such as extended surface loops or a decrease in the number of arginine residues or proline residues in loops were not observed. Because dominant features that could be related to the thermolability of the UDG from BMTU3346 cannot be identified, more subtle modifications of the conformation seem to be responsible for its thermolability.
Jaeger
S
S
Roche Diagnostics GmbH, Nonnenwald, Germany. stephan.jaeger@roche.com
Schmuck
R
R
Sobek
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D003081:Cold Climate; D045647:DNA Glycosylases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D051981:Uracil-DNA Glycosidase
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase