The Union of FBPase and SBPase
dTo aid in the naviagtion of the important experiement, each subject has been divided into three subheadings: 1. Definitions 2. Search Method 3. Conclusions

1. First Things First: Defining FBPase and SPBase
dfdfFructose-1,6-biosphophatase (FBPase) is the enzyme of the second bypass reaction of gluconeogenesis. The reaction is as follows:
dddddddddddddFructose 1,6-bisphosphate + H2O gluctose 6-phosphate + Pi

dddSedoheptulose-1,7-bisphosphatase (SBPase) is a key factor in carbon fixation of photosynthetic organisms.

2. Gene Searching
ddddThe goal of gene searching is to find genes coding for proteins of glycosomes (in this case FBPase) whereas the nearest homolog codes for a plant-like enzyme (in this case SBPase). The process is not complicated, but includes numerous steps. Each step brings the researcher closer to the exciting results: that trypanosomes are have unique metabolic machinery!

The process is as follows:
A search was initiated in the nucleic acid database GenBank using a query of bacterial and mammalian FBPases looking for gene sequences with the most identity of plant SBPases.
Based on the sequence of SBPase, two oligonucleotides were designed.
PCR amplification was performed using genomic DNA from T. brucei.
A major amplified 211-base pair product was cloned, sequenced (link), and used to screen the genomic library of T. brucei.
A maximum-likelihood tree was created using the program TREE-PUZZLE.

3. Conclusions: Coding for Both Functional Proteins!

The Striking Finding That Was Not Expected:
dddIn T. brucei, a gene with a complete ORF for a homolog of SBPase has been identified. Until this point, the enzyme has only been encountered in the chloroplasts of green algae and plants (Hannaert, 1068). The protein sequence has a targeting signal at its C-terminus that is suggestive of glycosomal localization. What is even more interesting is that when the protein sequences for FBPase and the newly-found SBPase are aligned with plant and algal SBPases and eukaryote FBPases, the residues essential for catalysis have been conserved in both trypanosome enzymes.
dddThis means…BOTH GENES STILL ENCODE FUNCTIONAL PROTEINS!

(Hannaert et al, 1069)

The Double Duty of Bifunctional Adolase

1. First Things First: Defining Aldolase
dfdfdffSo how exactly did the SBPase come to be as described in the above enzyme search? The only function for SBPase outside of a photosynthetic Calvin cycle is in a modified hexosemonophosphate pathway.
dfdfdfBifunctional aldolase is capable of forming both FBPase and SBPase by using either glyceraldehyde 3-phsphate or erythrose 4-phosphate as a substrate. Both use a condensation reaction with dihydroxyacetone phosphate. Until now, these adolases have only been found in chloroplasts.

2. Gene Searching
dddddThe T. brucei FBPase adolase sequence was compared with the complete SWISS-PROT database for homologous sequences using the BLAST engine and criteria. The best scoring 37 protein sequences were aligned using the program CLUSTALW. Below is the neighbor-joining tree.

3. Results
dddddThe trypanosome aldolase enzyme is most closely related to the enzymes of the rhodophyte alga and the chlorophyte algae. Trypanosoma aldolases appear to form a robust group with the chloroplast aldolases from algae. In addition, isoenzymes of the malaria parasite form a sister group of both the trypanosomatid and chloroplast aldolases.

dddddddddddddddd(Hannaert et al, 1069)

Hexoses in All The Wrong Places

1. First Things First: Defining Hexose-Monophosphate Pathway
dfdfdfdThe Hexose-Monophosphate Pathway or shunt is the major producer of NADPH used in reductive biosynthesis and catalyzes the interconversion of four, five, six and seven-carbon sugars. In mammals, this pathway is prominent in tissues actively carrying out biosynthesis of fatty acids and steroids from small precursor molecules, especially the mammary gland, adipose tissue, the adrenal cortex and the liver.
dfdfdfdfEnzymes of the pathway are found in the cytosol.
To view the full set of reactions of the pathway, click here.

2. Gene Searching
dfdfdfdfThe findings of FBPase, SBPase, and adolase relationships prompted scientists to investigate whether trypanosomatids contained other genes for which a plant or chloroplast affiliation could be inferred.

3. Results
dfdfdfffKrepinsky et al. recently reported that the first enzyme of the hexose-monophosphate pathway in trypanosomes, G6PDH, is most related to homologs found in the plant cytosol. In trypanosomes, this enzyme is present in both the cytosol and in glycosomes. The second enzyme from the hexose-monophosphate pathway, 6PGL, was also isolated and found to be present in glycosomes. The enzyme is of bacterial origin and clusters with cyanobacteria. The key link: Cyanobacteria are considered to be closely related to the precursors of all chloroplasts in plants.