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Journal of Bacteriology, November 2001, p. 6714-6716, Vol. 183, No. 22
The Rockefeller University, New York, New
York 100211; National Research Council
of Canada, Institute for Marine Biosciences, Halifax, Nova Scotia,
Canada B3H 4H72; and University of
Calgary, Faculty of Medicine, Department of Biochemistry and
Molecular Biology, Calgary, Alberta, Canada T2N
4N13
Received 14 May 2001/Accepted 24 August 2001
Inorganic pyrophosphate-dependent phosphofructokinase
(PPi-PFK) of the amitochondriate eukaryote
Mastigamoeba balamuthi was sequenced and showed about 60%
identity to PPi-PFKs from two eubacteria, Propionibacterium freudenreichii and Sinorhizobium
meliloti. These gene products represent a newly recognized
lineage of PFKs. All four lineages of group II PFKs, as defined by
phylogenetic analysis, contained both prokaryotic and eukaryotic
species, underlining the complex evolutionary history of this enzyme.
We have recently extended our
studies on glycolytic enzymes of parasitic amitochondriate eukaryotes
(20) to the free-living Mastigamoeba balamuthi
(ATCC 30984) (6) with the goal of comparing the metabolic
properties of anaerobic and microaerophilic eukaryotes with
dramatically different life styles. This species belongs to the
pelobionts, a group of amitochondriate amoeboflagellate protists of
uncertain evolutionary position (5, 25). We noted that the
sequence of its phosphofructokinase (PFK) showed unexpected characteristics, prompting us to revisit the taxonomic distribution and
relationships of various PFK types.
Type A PFK, an enzyme of the glycolytic pathway, phosphorylates
fructose 6-phosphate to fructose 1,6-bisphosphate. In most organisms,
ATP is the phosphoryl donor (ATP-PFK; EC 2.7.1.11) of the irreversible
reaction. A number of protists and plants and some eubacteria contain
reversible PFKs, which use inorganic pyrophosphate (PPi)
instead of ATP (PPi-PFK; EC 2.7.1.90). The assumed
significance of PPi as the phosphoryl donor is reflected in
an increase of the ATP yield during glycolysis (16, 26). This notion is supported by the predominant occurrence of
PPi-PFK in organisms living in hypoxic or anoxic
environments, which rely on anaerobic glycolysis (17).
The evolutionary history of PFK does not coincide with accepted notions
of organismic relationships and points to past gene duplications and
lateral gene transfers. Based primarily on sequence characteristics,
type A PFKs are currently assigned to three major groups (groups I, II,
and III) (22). Group II can be further subdivided into
four subgroups, which appear as robust clades in phylogenetic
reconstructions (we are using the tentative nomenclature proposed for
clades in group II [18]). Closely related organisms may
contain close homologs of PFK which use different phosphoryl donors,
indicating that enzyme specificity can change relatively easily
(2), a conclusion recently confirmed experimentally (7). Some organisms even contain members of two such
subgroups (8, 10-12).
Sequence of M. balamuthi PPi-PFK.
A
random clone of an M. balamuthi cDNA library (M. Müller et al., unpublished data), sequenced on both strands,
contained a G+C-rich (69.3%) open reading frame putatively encoding a
PPi-PFK of 410 amino acids with an
Mr of 44,200. The conceptual translation showed
that only 36 codons were used and that 98.2% of the nucleotides in the
third position of each codon were G or C. This skewed codon usage,
characteristic of protein-encoding genes of this organism (Müller
et al., unpublished data), and the presence of a typical eukaryotic
poly(A) tail support the origin of this message from authentic M. balamuthi DNA. The conceptual translation showed complete
colinearity and about 60% amino acid identity to the PPi-PFK of the gram-positive organism
Propionibacterium freudenreichii and to the putative
PPi-PFK of the Phylogenetic analysis.
Sequences of PPi-PFK
homologs were retrieved from the National Center for Biotechnology
Information protein database. The S. meliloti PFK sequence
was retrieved from the website of the corresponding genome project
(http://sequence.toulouse.inra.fr/meliloti.html) (3).
Sequence sampling for group II encompasses the whole diversity present
in the nonredundant National Center for Biotechnology Information
database. Sampling of group III was restricted to a few species to
provide an outgroup. Group I enzymes were not considered. The alignment
was performed using the CLUSTAL X program (23) and
adjusted visually. Phylogenetic reconstruction was performed with a
maximum-likelihood method (PROTML) (1) on 167 shared
amino-terminal amino acid positions. Bootstrap proportions were
calculated by a resampling of the estimated log likelihood (RELL)
values from the maximum-likelihood method (1). Neighbor joining and maximum-parsimony analyses revealed identical groups and
subgroups (data not shown).
Evolutionary relationships of group II and III PFK sequences.
The phylogenetic tree obtained shows five major clades (Fig.
1). Four clades belong to group II, and
one represents group III. The internal branches connecting these clades
are relatively long, and the branching pattern is supported by high
bootstrap values. The order of separation of lineages within these
clades has not been resolved, however. While this subdivision is based only on the easily aligned amino-terminal parts of the sequences, a
global comparison of the complete sequences and an analysis of the
lengths and positions of insertions and deletions result in an
identical subdivision (18).
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6714-6716.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Presence of Prokaryotic and Eukaryotic Species in All
Subgroups of the PPi-Dependent Group II
Phosphofructokinase Protein Family
ABSTRACT
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-proteobacterium, Sinorhizobium meliloti (3, 14). The M. balamuthi
sequence has been deposited in GenBank under accession number AAF70463.
View larger version (34K):
[in a new window]
FIG. 1.
Unrooted phylogenetic tree of group II and III
PPi-PFKs based on 167 shared amino-terminal residues
(between residues 2 and 204 in the P. freudenreichii
sequence). Taxon selection extended over the complete diversity present
in available databases. The arrow denotes a proposed gene duplication.
Close paralogs among chlamydiae, actinomycetales, and plants were
omitted for clarity. Database accession numbers follow the species
names. For the S. meliloti sequence, see
http://sequence.toulouse.inra.fr/meliloti.html. Eukaryotic species
names are in capital letters, and eubacterial species names are in
lowercase letters. A., Arabidopsis; M.t.,
Mycobacterium tuberculosis; S.c., Streptomyces
coelicolor. PPi specificity was established
biochemically for unmarked species; asterisks mark species not explored
biochemically, "ATP" indicates enzymes with ATP specificity, and
the question mark indicates a species for which the heterologously
expressed enzyme showed no PFK activity with either PPi or
ATP (9). For the long clade plant enzymes, is the
regulatory subunit and 
is the catalytic one. The maximum-likelihood
method with local rearrangements was utilized. Numbers at the nodes
give RELL bootstrap values (see text).
) and regulatory () subunits (4, 24). A similar duplication and functional change also occurred in group I, which contains the classical ATP-linked enzymes (21). The
functional significance of the chlamydial paralogs remains unknown.
The limited and peculiar taxonomic distribution of group II sequences
makes a coherent reconstruction of events leading to the observed
phenomena a daunting task. The relationships seen in the phylogenetic
reconstruction do not coincide with accepted organismic relationships.
One must account for the presence of both eubacteria and eukaryotes in
each of the four clades of group II PFK genes as well as for the
existence of sequences from the same organisms that fall into separate
clades. While both early gene duplications and subsequent differential
losses (15) and lateral gene transfers (13)
have probably contributed to the current picture, only a significantly
larger taxonomic sampling and functional characterization of the
proteins encoded will permit a convincing reconstruction of the
peculiar history of PPi-PFK homologs.
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ACKNOWLEDGMENTS |
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We thank Gordona Bothe (GATC-Biotechnology GMBH, Constance, Germany) for providing the S. meliloti sequence before its publication, Frederick Schuster (Brooklyn College, City University of New York, Brooklyn) for the M. balamuthi culture, Rama K. Singh and his team (NRC, Institute for Marine Biosciences, Halifax, Canada) for the DNA sequencing, Robert Kemp (The Chicago Medical School, North Chicago, Ill.) for suggestions and permission to refer to his paper before its publication, and William Martin (Heinrich-Heine Universität, Düsseldorf, Germany) and Lidya B. Sanchez and Dorothy V. Moore of the New York laboratory for comments on the manuscript.
This research was supported by grants to M.M. from the National Science Foundation (MCB9615659) and the National Institutes of Health (AI 11942).
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FOOTNOTES |
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* Corresponding author. Mailing address: The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8153. Fax: (212) 327-7974. E-mail: mmuller{at}mail.rockefeller.edu.
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Journal of Bacteriology, November 2001, p. 6714-6716, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6714-6716.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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