Identification of residues important for NAD+ binding by the Thermotoga maritima alpha-glucosidase AglA, a member of glycoside hydrolase family 4.
The NAD+-requiring enzymes of glycoside hydrolase family 4 (GHF4) contain a region with a conserved Gly-XXX-Gly-Ser (GXGS) motif near their N-termini that is reminiscent of the fingerprint region of the Rossmann fold, a conserved structural motif of classical nicotinamide nucleotide-binding proteins. The function of this putative NAD+-binding motif in the alpha-glucosidase AglA of Thermotoga maritima was probed by directed mutagenesis. The K(d) for NAD+ of the AglA mutants G10A, G12A and S13A was increased by about 300-, 5-, and 9-fold, respectively, while their K(m) for p-nitrophenyl-alpha-glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD+ binding by the glycosidases of GHF4.
Raasch
Carsten
C
Institut für Mikrobiologie und Genetik, Georg-August-Universität, Göttingen, Germany.
Armbrecht
Martin
M
Streit
Wolfgang
W
Höcker
Birte
B
Sträter
Norbert
N
Liebl
Wolfgang
W
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009243:NAD; D000520:alpha-Glucosidases