The actinomycete Amycolatopsis methanolicawas 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 PPi-PFK activity only in
members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and
3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-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. methanolicapyruvate kinase was
purified over 250-fold and characterized as an allosteric enzyme, sensitive to
inhibition by Pi 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.
INTRODUCTION
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 (Hodgson, 1994).Wehave initiated such
studies with the actinomycete Amycolatopsis methanolica (de Boer et al., 1990a),
belonging to the family Pseudonocardiaceae (Warwick et al., 1994) 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 (de
Boer et al., 1988; Dijkhuizen et al., 1992; Hazeu et al., 1983). Methanol oxidation via
formaldehyde and formate to carbon dioxide results in energy generation (Bystrykh et
al., 1993; Hazeu et al., 1983). Carbon assimilation starts by formaldehyde fixation via
the ribulose monophosphate (RuMP) cycle (de Boer et al., 1990; Hazeu et al., 1983). 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 (de Boer et al., 1990b), and various
enzymes also involved in the related pentose phosphate pathway (Fig. 1) (Dijkhuizen et
al., 1992).
The identity, properties, and regulation of enzymes involved in glucose and methanol
metabolism in A. methanolica were examined in this study.