Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha regulon responsible for fermentative dissimilation of glycerol and DHA, was purified 120-fold to a final specific activity of 10 ,umol x min-I x mg of protein-' at 30°C. The enzyme, a dimer of a 53,000 + 5,000-dalton polypeptide, is highly specific for DHA (Kn, ca. 4 ,uM). Glycerol is not a substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate. Antibodies raised against E. coli glycerol kinase cross-reacted with K. pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.Klebsiella pneumoniae has two different systems for the dissimilation of glycerol ( Fig. 1). With molecular oxygen or nitrate available as an exogenous electron acceptor, the glp regulon is used (14). In the absence of exogenous electron acceptors, the dha regulon is used (5,9,17,23,24,28). Metabolism of glycerol through the glp system is initiated by an ATP-dependent kinase. The product, sn-glycerol 3-phosphate (G3P), is oxidized to dihydroxyacetone phosphate by one of the flavin-linked dehydrogenases. Glycerol kinase is allosterically inhibited by fructose-1,6-diphosphate which prevents excessive phosphorylation of the substrate. (It was demonstrated in Es(herichia coli that accumulation of G3P results in growth stasis [2] and overabundance of dihydroxyacetone phosphate results in the lethal production of methylglyoxal [6,12].) Metabolism of glycerol through the dim system occurs by two parallel pathways, one serving for glycerol utilization, the other for the disposal of extra hydrogen. Utilization of glycerol is catalyzed by an NADlinked dehydrogenase (16,19) which converts the substrate to dihydroxyacetone (DHA). This product is then phosphorylated by an ATP-dependent kinase. Disposal of extra hydrogen is achieved by the sequential action of a B12dependent dehydratase and an NADH-linked oxidoreductase. Glycerol is first converted to 3-hydroxypropionaldehyde which then serves as a hydrogen sink for the regeneration of NAD. The resulting 1,3-propanediol (accounting for about 50% of the glycerol consumed) is excreted into the medium (3, 4, 20, 36). E. coli differs from K. pneuimoniae is possessing only the glp system. In this study, DHA kinase was purified from K. pnelumoniae for characterization and comparison with glycerol kinases from this organism and from E. (coli.Waters Associates, Milford, Mass., and was used on a Waters gradient HPLC system. Ultrogel ACA 34 was from LKB Instruments, Inc., Rockville, Md. Blue Sepharose CL-6B and protein standards for gradient polyacrylamide gel electrophoresis were purchased from Pharmacia Fine Chemicals, Piscataway, N.J. Special enzyme-grade ammonium sulfate was obtained from Schwarz/Mann, Orangeburg, N.Y. Rabbit muscle G3P dehydrogenase was obtained from Boehr...

The insect and plant pathogens within the fungal genus Verticillium showed enzymatic adaptation (production and regulation) directed to the degradation of some of the polymers found in the integument of their respective hosts. For example, the facultative plant pathogens (V. albo-atrum and V. dahliae) produced greater levels of cellulase and xylanase than the facultative insect pathogen (V. lecanii). Verticillium lecanii produced extracellular subtilisin-like protease when grown in insect cuticle medium but not in plant cell wall medium, while the plant pathogen V. albo-atrum showed a diminished regulatory component in the production of this enzyme. The opportunistic pathogens (V. fungicola and V. coccosporum) and the saprobic species (V. rexianum) were less specific in the production and regulation of several proteases as well as cellulases and xylanases. A dendrogram based on cluster analysis compiled from fungal API-ZYM profiles showed commonalties in a broad array of extracellular enzymes within a host-pathogen group (i.e. insect or plant pathogen). The opportunistic pathogens were dispersed throughout the dendrogram, suggestive of the diversity in type and expression of extracellular enzymes.Key words: extracellular enzymes, pathogenic fungi.