Cloning and characterization of Thermotoga maritima beta-glucuronidase.
The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.
Salleh
Hamzah M
HM
Protein Engineering Network of Centres of Excellence of Canada, Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Müllegger
Johannes
J
Reid
Stephen P
SP
Chan
Wing Y
WY
Hwang
Jiyoung
J
Warren
R Antony J
RA
Withers
Stephen G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005966:Glucuronidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
D005966:Glucuronidase