Next we look in detail at the differences in amino acid sequence between dihydropteroate synthase from sulfonamide susceptible meningococci and the corresponding resistant meningococci. We describe the mechanism as completely as possible and as a general example of resistance evolution in bacteria. A comparison of the amino acid sequences between susceptible and resistant enzymes, respectively, showed about 20 specific differences. Three of these affected amino acids, which have been identified in the same position in all known bacterial dihydropteroate synthases, indicating their role in the basic function of the enzyme (Fig. 1). One of those was Phe31 (phenylalanine; 31 is the consecutive number from the amino end of the peptide), which in the resistance enzyme is substituted for by Leu (leucine).

Fig1. Sulfonamide resistance in N. meningitidis. The effecto f specific amino acid changes on meningococcal resistance to sulfonamide. A stylized representation of the dihydropteroate synthase with the three amino acids that are important for resistance, marked by their sequence numbers. Above the protein symbol the amino acid configuration for susceptibility, Su^s is given, and below the protein symbol there is the configuration for resistance, Sur. To the right there are the values deter mined for the Michaelis constant (Km), the inhibition constant (Ki),and the MIC value for sulfonamide, in this case sulfathiazole. The different amino acid configurations show the effect of systematic amino acid exchanges by site-directed mutagenesis. See the text for further details.

This is functionally the same amino acid exchange that was involved in the spontaneous mutation to sulfonamide resistance in E. coli, described earlier in the chapter, where the corresponding phenylalanine has the consecutive number 28, which then gives Phe28Leu (phenylalanine in position 28 exchanged for leucine). In addition to Leu31, the resistance enzyme in meningococci also has Ser84 and Cys194 as exchanged amino acids. By site-directed mutagenesis experiments in vitro, these amino acid exchanges were systematically restored to the amino acid pattern of the susceptible enzyme. When Leu31forresistance was changed to Phe31 of the susceptible enzyme, the resistance decreased 25-fold; that is, the (minimum inhibitory concentration (MIC) value) for sulfonamide decreased from 0.5 mM to less than 0.02 mM. When, correspondingly, the Cys194 of resistance was exchanged for the Gly194 of susceptibility, the MIC value for sulfonamide decreased from 0.5 mM to 0.12 mM. Both of these amino acid changes are thus closely related to resistance. It could be addedthatbothLeu31 andCys194arelocated in highly conserved areas of the enzyme peptide; that is, they are very similar, if not identical, among all known bacterial dihydropteroate synthases, which in turn ought to mean that they are involved in the catalytic function of the enzyme. Finally, when Ser84 for resistance was changed to Pro84 corresponding to susceptibility, no effect on MIC could be observed.

Quantitative measurements of enzyme kinetics were per formed to further characterize sulfonamide resistance. In these experiments, the artificially mutated meningococcal gene for dihydropteroate synthase ,borne on a small plasmid, was allowed to express itself in a laboratory strain of E. coli in which the chromosomal gene for dihydropteroate synthase had been inactivated by a partial deletion. Extracts from such a system would thus contain only meningococcal enzyme, and sulfonamide effects on mutated forms of this enzyme could then be measured specifically and precisely. It was shown that the Ki value for sulfonamide (in this case, sulfathiazol), which is a quantitative measure of the affinity of the inhibitor for the enzyme, showed good correlation with the MIC values assessed (Fig. 1). The amino acid exchange of resistance Leu31 to susceptibility 31Phe caused an almost 400-fold decrease in the Ki value for sulfonamide. At the same time, a sixfold decrease in the Km value for the normal substrate of p-aminobenzoic acid could be observed. Also, changing the Cys194 of resistance to Gly194 of susceptibility resulted in a substantial decrease in Ki, whereas changing resistance Ser84 to susceptibility 84Pro increased the Ki value for sulfonamide threefold and also increased the Km value for p-aminobenzoic acid twofold. The resistance Ser84 can be regarded as a compensatory amino acid exchange, stabilizing the resistance enzyme to show the same efficiency as that of the original sensitive enzyme.

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