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The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulosebinding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50°C versus 60-65°C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported -on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A.

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An enzyme with a specificity that has not been described previously, D-arabitol-phosphate dehydrogenase (APDH), has been purified from cell lysate of Enterococcus avium. SDS/PAGE indicated that the enzyme had a molecular mass of 41+/-2 kDa, whereas a molecular mass of 160+/-5 kDa was observed under non-denaturing conditions, implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have a narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into xylulose 5-phosphate and ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD(+) and NADP(+) were accepted as cofactors. Based on the partial protein sequences, the APDH gene was cloned. Homology comparisons place APDH within the medium-range dehydrogenase family. Unlike most members of this family, APDH requires Mn(2+) but no Zn(2+) for enzymic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system. Both biochemical evidence and protein sequence homology comparisons indicate that similar enzymes are widespread among the Gram-positive bacteria. Their apparent biological role is to participate in arabitol catabolism via the 'arabitol phosphate route', similar to the ribitol and xylitol catabolic routes described previously.

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