Sedoheptulose-1,7-bisphosphatase
and fructose-1,6-bisphosphatase
The most striking observation of a
typical plant-like gene we identified in Trypanosoma brucei
contains a complete open-reading frame encoding a homologue of
sedoheptulose-1,7-bisphosphatase (SBPase), an enzyme typical for the
Calvin cycle of photosynthetic organisms and further only encountered
in the chloroplasts of green algae and plants (Martin et al, 1996).
Alignment
of its predicted amino-acid sequence
with other SBPases from both plants and the green alga
Chlamydomonas reinhardtii and with sequences of the related
gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) from both
bacteria and eukaryotes indicates that all residues essential for
catalysis have been conserved in the trypanosome SBPase. This
suggests that the trypanosome gene still encodes a functional enzyme.
Not only does the trypanosome contain a typical chloroplast enzyme
but, as do only plants, it contains both an SBPase and an FBPase.
The
phylogenetic tree
shows that the closest affiliation of
the trypanosome enzyme is with that of Chlamydomonas. While
the trypanosome SBPase robustly clusters with the plant SBPases, the
T.brucei FBPase branches at the bottom of the eukaryotic clade
of FBPases, suggesting a truly protist nature for this enzyme.
Further details of the analysis
The T. brucei SBPase was compared with the SwissProt database indexed at European Bioinformatics Institute (EBI, Hinxton UK) on 28 July 2001 and having 99162 entries, using the NCBI BLASTP program and the BLOSUM 62 matrix (http://www2.ebi.ac.uk/blastall/). Click here to inspect the BLASTP output file.
The best E values (1e-56 to 7e-54) were scored with the three available plant SBPases and with the one of the chlorophyte alga Chlamydomonas (2e-48). All other homologues were those of bifunctional FBP/SBPases and monofunctional FBPases, which gave much poorer scores, ranging from 5e-25 to 2e-11.
The T. brucei SBPase sequence shared about 40% positional identity with the plant SBPase sequences, while they shared 30% positional identity, or less, with the FBPases. All 44 sequences available from the SwissProt database were aligned using the "RunDBClustalW" option in the BLASTP output. The ClustalW alignment is availabe here for inspection. The top line represents the trypanosome SBPase sequence. Please note the presence of a C-terminal peroxisome targeting signal (-SKL) in the T. brucei protein.
A similar analysis was carried out with the T. brucei FBPase . Click here to inspect the resulting BLASTP output file for FBPases. All homologous sequences with E values ranging from 4e-82 for Brassica namus (rape) FBPase to 2e-18 for Chlamydomonas SBPase were selected for the creation of a multiple alignment. Please note the presence of a C-terminal PTS1 targeting sequence (-SKL) in the T. brucei FBPase (first sequence in the alignment) as well as a putative N-terminally located PTS2 targeting signal (MPDYSRVPSTLMQFLMHNQPKG). This strongly suggests that both FBPase and SBPase from T. brucei are targeted to the glycosomes.
The two alignments, which essentially differed by the presence or absence of either the T. brucei FBPase or SBPase, respectively, were saved to disk, imported into ClustalX and then combined using the profile alignment option of ClustalX, such that both the T. brucei FBPase and SBPase were included in the same alignment. Duplicate sequences were removed. The resulting alignment was inspected for correctness and then used for the construction of phylogenetic trees. The "Tree" option in ClustalX with "exclusion of regions with gaps" and with "correction for multiple substitutions" was used for the creation of a bootstrapped (1000 samplings) neighbor-joining tree. For the creation of a protein maximum likelihood tree the alignment was first converted to Phylip format and all positions with gaps removed. The alignment contained 44 sequences and 260 sites.
An uncorrected distance matrix indicated that the T. brucei SBPase differed by 62 - 63% from the SBPases from plants and the chlorophyte alga, while it differed by 70% from the T. brucei FBPase, and 71-75% from the other FBPases.
The dataset contained a strong phylogenetic signal (only 3% of star-like quartets) when analysed with maximum likelihood mapping as implemented in Puzzle version 4.0.1. The ML tree was created with the program Puzzle, using the JTT model and 1000 puzzle steps. The resulting NJ and ML tree files were imported into the program NJTree for editing and annotation. T. brucei SBPase clustered robustly (100 % support) with the SBPases from plants in both NJ and ML trees. The maximum parsimony tree gave a highly similar topology also with strong bootstrap support (92%) for the clustering of the T. brucei SBPase with the other SBPases. Four-cluster likelihood mapping also gave strong support (100%) for a clustering of T. brucei SBPase with its homologues from chloroplasts.
Conclusion:
All methods (distance matrix, neighbor joining, maximum likelihood,
maximum parsimony and likelihood mapping) used in this analysis gave
a very strong support for a clustering of the T. brucei SBPase
sequence with that of the chloroplast sequences. Moreover, the fact
that T. brucei has a gene for both an SBPase and a FBPase,
which are only distantly related, supports the idea that one of these
two genes must have entered the trypanosome ancestor by an event of
horizontal gene transfer.
What is the function of these two proteins
in T. brucei glycosomes?
The FBPase is supposed to be involved in gluconeogenesis, a
pathway not operational in the bloodstream form due to the abundance
of glucose in the host's bloodstream. We expect the pathway to be
operational in other life-cycle stages of this parasite. Although we
have not yet been able to detect the FBPase activity in insect
stages, there are indications that the enzyme must be functional.
Insect stages utilize amino acids rather than carbohydrates as energy
substrates and are able to grow in the absence of carbohydrates. Yet
these cells synthesize glycoproteins and glycolipids for
incorporation in their plasma membrane.
The SBPase releases the C1 phosphate from
sedoheptulose 1,7-bisphosphate (S17BP), an intermediate of the
photosynthetic Calvin cycle, so forming sedoheptulose 7-phosphate, an
intermediate of both the Calvin cycle and the hexose-monophosphate
pathway. The functioning of the SBPase inside glycosomes can be
explainded by the presence inside glycosomes of a
fructose-bisphosphate aldolase of chloroplast affiliation. All
chloroplast aldolases have a wide substrate specificity allowing them
to utilise either dihydroxyacetone-phosphate and
glyceraldehydephosphate to form fructose 1,6 -bisphosphate or
dihydroxyacetone-phosphate and erythrose 4-phosphate to form S17BP.
Enzymes: 1, glucose-6-phosphate
dehydrogenase; 2, 6-phosphogluconolactonase; 3,
6-phosphoglucononate dehydrogenase; 4, ribulose-5-phosphate
3-epimerase; 5, ribulose-5-phosphate isomerase; 6,
transketolase; 7, transaldolase; 8, transketolase; 9,
triose-phosphate isomerase; 10, aldolase; 11,
sedoheptulose-1,7-bisphosphatase
