A. Husain et al. / Molecular & Biochemical Parasitology 170 (2010) 100–104
103
Table 2
l-glycine can be converted to pyruvate via l-serine by the action
of SHMT and TD. The later route of threonine catabolism does not
operate in Entamoeba as a gene encoding SHMT is absent [7]. In
T. vaginalis, which possesses TD, l-threonine and l-serine are also
summary, we have characterized TD, one of the key enzymes that
allow utilization of amino acids as energy source [22,23], from E.
histolytica. Our findings should help to understand the significance
of the amino acid catabolism for energy generation in E. histolytica
[24].
Inhibition of recombinant EhTD1 by keto-acids.
%Inhibition
Without AMP
With 5 mM AMP
Pyruvate
Glyoxylate
2-Oxobutyrate
45.3 1.7
54.1 1.2
49.3 1.1
44.6 0.2
49.6 2.7
43.2 3.8
TD activity was measured spectrophotometrically at 310 nm as described previously
[15]. Effects of 20 mM of various keto acids (pyruvate, 2-oxobutyrate or glyoxylate)
on the activity of rEhTD1 in the presence or absence of AMP (5 mM). TD activity is
keto acid and AMP. All values are expressed as a mean S.E. of triplicates.
Acknowledgements
We thank Kumiko Nakada-Tsukui, Fumika Mi-ichi, and all other
members of our laboratory for the technical assistance and valu-
able discussions. This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan to T.N. (18GS0314, 18050006,
18073001), a grant for research on emerging and re-emerging
infectious diseases from the Ministry of Health, Labour and Wel-
fare of Japan (H20-Shinkosaiko-016), and a grant for research to
promote the development of anti-AIDS pharmaceuticals from the
Japan Health Sciences Foundation to T.N.
(Table 1). Therefore, l-cysteine likely plays a role in the regulation of
logical concentrations. We also studied the effect of ATP, ADP, AMP,
and CMP on the activity of rEhTD1. All of these nucleotides have
been shown to be allosteric regulators of catabolic TDs from vari-
ous sources [4]. rEhTD1 was inhibited by ATP in a dose-dependent
manner (data not shown). It showed approximately 50% inhibition
plants and animals [4,15], rEhTD1 was insensitive to the allosteric
activation by ADP, AMP, and CMP (data not shown). The AMP/CMP
binding site of EhTD1 has three substitutions of Arg-53 to Lys-69,
Asp-119 to Gly-135, and Gln-275 to Glu-291 (Fig. 2, “D”). The guani-
dium group of Arg-53 forms a hydrogen bond with the phosphate
group of AMP or CMP. The side-chain oxygen atom of Asp-119 is
hydrogen bonded to the N4 atom of the adenine moiety of AMP
or the N3 atom of the cytosine moiety of CMP, whereas the side-
chain oxygen atom of Gln-275 is involved in the hydrogen bonding
with the N6 atom of the adenine moiety of AMP or the O2 atom of
the cytosine moiety of CMP [4]. Absence of these hydrogen bonds
might contribute to the lack of AMP or CMP binding to the enzyme.
Allosteric activation by valine and inhibition by isoleucine were
described for biosynthetic TDs [3]. However, in accordance with
the catabolic nature, rEhTD1 was neither activated by valine nor
inhibited by isoleucine (data not shown). We also investigated the
effect of various catabolites on the activity of rEhTD1 in both the
presence and absence of AMP. rEhTD1 was markedly inhibited by
both the end products, i.e. pyruvate and 2-oxobutyrate, and also by
glyoxylate (Table 2). Inhibition by these keto acids was not reversed
other sources. This is in accordance with the lack of allosteric acti-
vation of EhTD1 by AMP. Inhibition of catabolic TDs by the keto
mented [15]. Altogether, these findings indicate that the regulatory
mechanisms of EhTD1 are distinct from that of bacterial, plant, and
animal counterparts.
tyostelium discoideum [19]. D. discoideum has both catabolic and
biosynthetic TDs, and during the development of the vegetative to
pseudoplasmodial stage, biosynthetic TD activity decreases while
catabolic TD is upregulated [19]. Similarly, TD is also developmen-
tally regulated in Entamoeba. Our transcriptome study showed that
an E. invadens homologue of EhTD1 gene is down regulated by 8 fold
during the transition of trophozoites to the dormant cyst stage,
suggesting its role in proliferation (Escueta et al., unpublished).
In Trypanosoma brucei, which lacks TD, l-threonine is metabo-
lized using the alternative aminoacetone pathway, which involves
a mitochondrial threonine dehydrogenase and aminoacetone syn-
thase. This route is not operational in E. histolytica and Leishmania
major [20]. L. major has two pathways to metabolize l-threonine.
l-threonine is converted to 2-oxobutyrate by TD, and then oxidized
to succinyl CoA. Alternatively, l-threonine can be converted to l-
glycine by serine hydroxymethyltransferase (SHMT). The resulting
References
[1] Umbarger HE. Threonine deaminases. Adv Enzymol 1973;37:349–95.
[2] Phillips AT, Wood WA. The mechanism of action of 5ꢀ-adenylic acid-activated
threonine dehydratase. J Biol Chem 1965;240:4703–9.
[3] Umbarger HE. Evidence for a negative feedback mechanism in the biosynthesis
of isoleucine. Science 1956;123:848.
[4] Simanshu DK, Savithri HS, Murthy MRN. Crystal structures of Salmonella
typhimurium biodegradative threonine deaminase and its complex with CMP
provide structural insights into ligand-induced oligomerization and enzyme
activation. J Biol Chem 2006;281:39630–41.
[5] World Health Organization/Pan American Health Organization Report.
A
consultation with experts on amebiasis. Epidemiological Bulletin/PAHO
1997;18:13–4.
[6] Müller M. Enzymes and compartmentation of core energy metabolism of
anaerobic protists—a special case in eukaryotic evolution. In: Coombs GH, Vick-
erman K, Sleigh MA, Warren A, editors. Evolutionary Relationships Among
Protozoa. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1998.
p. 109–32.
[7] Loftus B, Anderson I, Davies R, et al. The genome of the protist parasite Enta-
moeba histolytica. Nature 2005;433:865–8.
[8] Zuo X, Coombs GH. Amino acid consumption by the parasitic, amoeboid pro-
tists Entamoeba histolytica and E. invadens. FEMS Microbiol Lett 1995;130:
253–8.
[9] Samarawickrema NA, Brown DM, Upcroft JA, Thammapalerd N, Upcroft P.
Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreduc-
tase in mechanisms of Metronidazole resistance in Entamoeba histolytica. J
Antimicrobial Chemother 1997;40:833–40.
[10] Reeves RE, Warren LG, Susskind B, Lo HS. An energy-conserving pyruvate-to-
acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate
thiokinase. J Biol Chem 1977;252:726–31.
[11] Takeuchi T, Weinbatch EC, Gottlieb M, Diamond LS. Mechanism of l-
serine oxidation in Entamoeba histolytica. Comp Biochem Physiol B 1979;62:
281–5.
[12] Diamond LS, Harlow DR, Cunnick CC. A new medium for the axenic cultiva-
tion of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg
1978;72:431–2.
[13] Nozaki T, Asai T, Kobayashi S, et al. Molecular cloning and characterization
of the genes encoding two isoforms of cysteine synthase in the enteric pro-
tozoan parasite, Entamoeba histolytica. Mol Biochem Parasitol 1998;97:33–
44.
[14] Soda K. Microdetermination of d-amino acids and d-amino acid oxidase activ-
ity with 3-methyl-2-benzothiazolone hydrazone hydrochloride. Anal Biochem
1968;25:228–35.
[15] Shizuta Y, Kurosawa A, Inoue K, Tanabe T, Hayaishi O. Regulation of
biodegradative threonine deaminase. I. Allosteric inhibition of the enzyme by
a reaction product and its reversal by adenosin 5ꢀ-monophosphate. J Biol Chem
1973;248(2):512–20.
[16] Saito N, Robert M, Kochi H, et al. Metabolite profiling reveals YihU as a
novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde
metabolism in Escherichia coli. J Biol Chem 2009;284:16442–51.
[17] Bakker Grunwald T, Martin JB, Klein G. Characterization of glycogen and amino
acid pool of Entamoeba histolytica by 13C-NMR spectroscopy. J Eukaryot Micro-
biol 1995;42:346–9.