Doležal, P., Tachezy J., Proost, P., Hrdý I. (2002) Malic enzymes of Trichomonas
vaginalis: Purification, function and phylogeny. Abstract in The Journal of Eukaryotic
Microbiology 49 (2): 7 A-8A ISSN 1066-5234, IF 1,739
ABSTRACT. Trichomonas vaginalis possesses two types of malic enzymes: hydrogenosomal
NAD(P)-specific and cytosolic NADP-specific malic enzyme. Hydrogenosomal type
is a homotetramer of the 60 kDa subunits. It utilizes NAD+ preferentially to NADP+
and supplies hydrogenosome with pyruvate for further catabolic processes. In this
study we focused on NADP-specific malic enzyme from the cytosol. One of the several
present isoforms was purified to homogeneity by liquid chromatography. Molecular
weight determination of the native purified isoform suggested an unusual dimeric
arrangement of the 42 kDa subunits. The enzyme utilizes exclusively NADP+ (Km,
2,8 µM) with pH optimum between 7,5 -8,5. The main function of the cytosolic malic
enzyme is probably production of NADPH which is then utilized in the formation
of the major glycolytic end product, glycerol. Moreover, together with the consecutive
action of phosphoenolpyruvate carboxykinase and malate dehydrogenase the cytosolic
malic enzyme forms a pathway that bypasses pyruvate kinase reaction and transfers
reducing equivalents from NADH to NADP+. We cloned and sequenced the complete
gene of NADP-specific malic enzyme. The coding region is preceded by initiator
element while 3´-flanking region contains polyadenylation signal, thus corresponding
to known arrangement of transcription unit in T. vaginalis. Amino acid sequence
comparisons with homologous proteins revealed eubacterial features of cytosolic
malic enzyme and indicated totally different evolutionary origin of both types
of T. vaginalis malic enzymes. Hydrogenosomal type appears to be one of the most
divergent eukaryotic malic enzyme. Apart from their obvious role in metabolism
of T. vaginalis, several studies have described specific extracellular release
of the malic enzymes and suggested their adhesive function. To verify this hypothesis,
we analyzed the proteins excreted by T. vaginalis into Doran medium. In addition
to described secretion of both malic enzymes we detected other cytosolic (LDH,
MDH) and hydrogenosomal (STK) proteins as well. We also examined effect of T.
vaginalis secreted proteases on the activity of MDH, LDH and malic enzymes. In
cell-free supernatants obtained after one-hour cell incubation both malic enzyme
remained active over four hours while activities of MDH and LDH were undetectable
after 30 minutes. We propose that extracellular localization of malic enzymes
does not indicate their adhesive function but rather reflects unspecific exocytosis
of cell content in combination with resistance of malic enzymes to proteolytic
degradation.