Fig. 2.

The d-xylose binding site of XylFII. (A) Interaction of amino acid residues with d-xylose in the binding site. The bound d-xylose (cyan) is depicted as a ball-and-stick model. The side chains of the residues involved in the specific binding of d-xylose are shown in a zoom-in view. (B) Binding affinities of d-xylose to XylFII-LytSN and mutants measured using ITC. Binding curves of XylFII-LytSN (WT) and mutants XylFII^Y28A-LytSN (Y28A), XylFII^W29A-LytSN (W29A), and XylFII^D103A-LytSN (D103A) measured by ITC are shown in blue, red, green, and purple, respectively. (C) Growth of C. beijerinckii affected by the d-xylose binding site mutations. The growth rates were monitored by transforming XylFII (WT), empty pXY1 vector (EV), XylFII^Y28A (Y28A), XylFII^W29A (W29A), XylFII^D103A (D103A), or XylFII^R155A (R155A) genes to the xylFII mutant strain and growing them in YP2 medium with d-xylose as the sole carbon source. The bars indicate the SDs of three independent experiments. (D) d-xylose consumption of curve of C. beijerinckii affected by the d-xylose binding site mutations. The residual d-xylose in the medium were monitored by transforming XylFII (WT), empty pXY1 vector (EV), XylFII^Y28A (Y28A), XylFII^W29A (W29A), XylFII^D103A (D103A), or XylFII^R155A (R155A) genes to the xylFII mutant strain and growing them in YP2 medium with d-xylose as the sole carbon source. The bars indicate the SDs of three independent experiments. (E) Expression levels of genes affected by the d-xylose binding site mutations. The expression of xylFII, the downstream target d-xylose ABC transporter xylFGH genes, and a gene encoding pullulanase (control; CK) were measured by qRT-PCR. Gene expression levels are represented as fold differences normalized to WT. The error bars indicate the SDs of three independent experiments.