Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction. Biochemical and genetical analyses show that dihydroxyacetone and D-glyceraldehyde are converted into glycerol and then catabolized by the same pathway. D-Glyceraldehyde can be reduced by NADP+-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PP,-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PP,-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PP,-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P, and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.Actinomycetes are important bacterial producers of secondary metabolites. There is a strong interest in the genetics of secondary-metabolite biosynthesis, with most studies concentrating on these pathways and their control. Many secondary metabolites are initially derived from intermediates of the central pathways of primary metabolism. Little is currently known, however, about the enzymes and regulation of, for instance, glucose metabolism in actinomycetes. This is mostly because of a general lack of physiological studies on primary metabolism in actinomycetes (21). We have initiated such studies with the actinomycete Amycolatopsis methanolica (8), belonging to the family Pseudonocardiaceae (42), which includes many species producing bioactive compounds, e.g., the antibiotics rifamycin and erythromycin. A. methanolica is one of the few methanol-utilizing gram-positive bacteria known (10,12,17). Methanol oxidation via formaldehyde and formate to carbon dioxide results in energy generation (5,17). Carbon assimilation starts by formaldehyde fixation via the ribulose monophosphate (RuMP) cycle (9,17). This cycle involves the specific enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), the glycolytic enzymes 6-phosphofructokinase (PFK) and fructose-1,6-bisphosphate (FBP) aldolase (9), and various enzymes also involved in the related pentose phosphate pathway (Fig. 1) (12).The identity, properties, and regulation of enzymes involved in glucose and methanol metabolism in A. methanolica were examined in this study. MATERUILS AND METHODSMicroorganisms and cultivation. Wild-type A. methanolica NCIB 11946, its maintenance, and the procedures followed for cultivation in batch cultures, harvesting of cells, and growth measurements have been described previously (8)(9)(10)(11)

Glycerol dehydrogenase, NADP+-specific (EC 1.1.1.72), was purified from mycelium of AspergiZZus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an M, of approximately 38000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. n&er enzyme at pH 6-0, whereas inhibition by NADP+ was less (Ki = 45 ~L M vs 13 p~). It is proposed that both enzymes catalyse in #iuo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.

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The chromosome VIII translocation breakpoint of the areB-404 translocation, selected for its ability to activate the cryptic nitrogen metabolism regulatory gene areB, and the mutation glcD-100 both lead to loss of mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase in Aspergillus nidulans. These two lesions therefore define glcD, a second gene (in addition to glcB) where mutation can result in loss of this enzyme. The glcD gene has been localised to a centromere-proximal region of the right arm of chromosome VIII. Although all six known areB-activating mutations involve chromosomal rearrangements and presumably therefore gene fusions, areB-404 is the first such rearrangement where the gene involved in an areB fusion has been identified.