The unique glycoside hydrolase family 77 amylomaltase from Borrelia burgdorferi with only catalytic triad conserved.
Glycoside hydrolase family 77 (GH77) contains prokaryotic amylomaltases and plant-disproportionating enzymes (both possessing the 4-alpha-glucanotransferase activity; EC 2.4.1.25). Together with GH13 and GH70, it forms the clan GH-H, known as the alpha-amylase family. Bioinformatics analysis revealed that the putative GH77 amylomaltase (MalQ) from the Lyme disease spirochaete Borrelia burgdorferi genome (BB0166) contains several amino acid substitutions in the positions that are important and conserved in all GH77 amylomaltases. The most important mutation concerned the functionally important arginine positioned two residues before the catalytic nucleophile that is replaced by lysine in B. burgdorferi MalQ. Similar remarkable substitutions were found in two other putative GH77 amylomaltases from related borreliae. In order to confirm the exclusive sequence features and to verify the eventual enzymatic activity, the malQ gene from B. burgdorferi was amplified using PCR. A c. 1.5-kb amplified DNA fragment was sequenced, cloned and expressed in Escherichia coli, and the resulting recombinant protein was preliminarily characterized for its activity towards glucose (G1) and a series of malto-oligosaccharides (G2-G7). This study confirmed that the remarkable substitution of the arginine really exists and the GH77 MalQ protein from B. burgdorferi is a functional amylomaltase because it is able to hydrolyse the malto-oligosaccharides as well as to form their longer transglycosylation products.
Godány
Andrej
A
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
Vidová
Barbora
B
Janecek
Stefan
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D025065:Borrelia burgdorferi / Q000201:enzymology* / Q000235:genetics*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; C020569:4 alpha-glucanotransferase; D005947:Glucose