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The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64°C and an exothermic event centered near 75°C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications. Proteins 2003;50:341–354. © 2002 Wiley-Liss, Inc.

The signal peptide (sp) in Streptomyces lividansxylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine‐Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividansharboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti‐xylanase antibodies followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse‐chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividansrapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.

The atomic-resolution structure of a xylobiose-derived isofagomine lactam in complex with the xylanase Xyn10A from Streptomyces lividansreveals that the lactam is bound to the enzyme as the amide tautomer, with “reversed” protonation-states for nucleophile and acid–base.