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Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.

International audience; Mycobacterium fallax (M. fallax) is naturally sensitive to many beta-lactam antibiotics (MIC < 2 microg/ml) and devoid of beta-lactamase activity. In this paper, we show that the production of the beta-lactamase of Mycobacterium fortuitum by M. fallax significantly increased the MIC values for good substrates of the enzyme, whereas the potency of poor substrates or transient inactivators was not modified. The rates of diffusion of beta-lactams through the mycolic acid layer were low, but for all studied compounds the half-equilibration times were such that they would only marginally affect the MIC values in the absence of beta-lactamase production. These results emphasize the importance of enzymatic degradation as a major factor in the resistance of mycobacteria to penicillins.

The beta-lactamase of Mycobacterium smegmatis mc(2)155 has been purified to protein homogeneity. Its N-terminal sequence and catalytic properties are similar to those of the beta-lactamase produced by Mycobacterium fortuitum D316 and establish this new enzyme as a member of molecular class A.

Sixteen different compounds usually considered beta-lactamase stable or representing potential beta-lactam inhibitors and inactivators were tested against the beta-lactamase produced by Mycobacterium fortuitum. The compounds exhibiting the most interesting properties were BRL42715, which was by far the best inactivator, and CGP31608 and ceftazidime, which were not recognized by the enzyme. These compounds thus exhibited adequate properties for fighting mycobacterial infections. Although cloxacillin, dicloxacillin, cefoxitin, and CP65207-2 exhibited poor inhibitory efficiency against the enzyme, they were also rather poor substrates and might be considered potential antimycobacterial agents. By contrast, CGP31523A and ceftamet were good substrates.