The phosphocarrier HPr (heat stable protein) of Staphylococcus carnosus was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supposed to be part of the active centre of the protein. Three residues which are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: Arg17 was changed to His (17RH) and Pro18 and Glu84 were changed into Ala (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (delta 83). The wild type gene and all mutant genes were overexpressed and the gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All five mutant HPrs had native conformations. The ATP-dependent HPr kinase can phosphorylate all HPr derivatives at Ser46. The PTS activity of the amino-terminal HPr mutant proteins 17RH and 18PA was different compared to wild type HPr. In contrast, the carboxy-terminal mutant HPrs possessed a similar enzyme activity to the wild type HPr. The 17RH and 18PA HPrs with substitution near the active centre His15 showed a very slow phosphorylation by enzyme I but the further transfer of the phosphoryl group to enzyme III was also strongly inhibited. The enzyme activity of the HPr 17RH was significantly improved at low pH. NMR pH-titration experiments showed that Arg17 is not responsible for the low pKa of the active centre His15 but this positively charged residue is essential in this position for the HPr activity.

New information about the proteins of the phosphotransferase system (PTS) and of phosphoglycosidases of homofermentative lactic acid bacteria and related species is presented. Tertiary structures were elucidated from soluble PTS components. They help to understand regulatory processes and PTS function in lactic acid bacteria. A tertiary structure of a membrane-bound enzyme II is still not available, but expression of Gram-positive genes encoding enzymes II can be achieved in Escherichia coli and enables the development of effective isolation procedures which are necessary for crystallization experiments. Considerable progress was made in analysing the functions of structural genes which are in close vicinity of the genes encoding the sugar-specific PTS components, such as the genes encoding the tagatose-6-P pathway and the 6-phospho-beta-glycosidases. These phosphoglycosidases belong to a subfamily of the beta-glycosidase family I among about 300 different glycosidases. The active site nucleophile was recently identified to be Glu 358 in Agrobacterium beta-glucosidase. This corresponds to Glu 375 in staphylococcal and lactococcal 6-phospho-beta-galactosidase. This enzyme is inactivated by mutating Glu 375 to Gln. Diffracting crystals of the lactococcal 6-P-beta-galactosidase allow the elucidation of its tertiary structure which helps to derive the structures for the entire glycosidase family 1. In addition, a fusion protein with 6-phospho-beta-galactosidase and staphylococcal protein A was constructed.

Rilopirox is a synthetic, fungicidal antimycotic agent with hydrophobic characteristics. Its chemical name is 6-[4-(4-chlorophenoxy)-phenoxymethyl]-1-hydroxy-4-methyl-2-pyridone and it has a molecular weight of 357.79. Rilopirox is very soluble in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) but poorly soluble in water. The amount of antimycotic agent remaining in the solution is dependent on the final concentration of the solvent and the amount of rilopirox used. Complexometric studies show that rilopirox has a high affinity for iron ions [unpubl. data]. Catalase, an iron-containing enzyme, is inhibited by the chelating agent rilopirox. Studies on yeast mitochondria and submitochondrial particles show that rilopirox inhibits the respiratory chain. Complex I (NADH-ubiquinone oxidoreductase) contains iron-sulfur proteins and is the main system which is inhibited.

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