Description:
<jats:p>The NAD<jats:sup>+</jats:sup>‐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<jats:sup>+</jats:sup>‐binding motif in the α‐glucosidase AglA of<jats:italic>Thermotoga maritima</jats:italic>was probed by directed mutagenesis. The<jats:italic>K</jats:italic><jats:sub>d</jats:sub>for NAD<jats:sup>+</jats:sup>of the AglA mutants G10A, G12A and S13A was increased by about 300‐, 5‐, and 9‐fold, respectively, while their<jats:italic>K</jats:italic><jats:sub>m</jats:sub>for<jats:italic>p</jats:italic>‐nitrophenyl‐α‐glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD<jats:sup>+</jats:sup>binding by the glycosidases of GHF4.</jats:p>