Functional analysis of the 5' untranslated region of the phosphoglutamase 2 transcript in Plasmodium falciparum.

Plasmodium falciparum transcripts contain long untranslated regions (UTR), with some of the longest in any eukaryote that uses monocistronic transcription. Owing to the extreme AT nucleotide bias within the intergenic regions that encode these UTR, attempts to characterise how they are apportioned over genes and to describe their contribution to the absolute and temporal control of gene expression have been limited. Here we describe a study using a typical house-keeping gene that encodes phosphoglutamase 2 (PFD0660w), whose expression is subject to developmentally linked control during intraerythrocytic development. We show that deletion of a significant proportion (80%) of the predicted 5' UTR has no apparent effect on the developmentally linked expression of a luciferase reporter cassette. Further, serial deletions reveal that whilst the absolute level of transcription is unaffected when up to 50% of the predicted 5' UTR is removed, the subsequent efficiency of translation is affected. These data provide key insights into the interplay of transcriptional and post-transcriptional mechanisms in the control of gene expression in this important human pathogen.

Crystal structures of Leishmania mexicana phosphoglycerate mutase suggest a one-metal mechanism and a new enzyme subclass.

The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-A resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co(2+) are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co(2+) binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.

Unexpected catalytic site variation in phosphoprotein phosphatase homologues of cofactor-dependent phosphoglycerate mutase.

The cofactor-dependent phosphoglycerate mutase (dPGM) superfamily contains, besides mutases, a variety of phosphatases, both broadly and narrowly substrate-specific. Distant dPGM homologues, conspicuously abundant in microbial genomes, represent a challenge for functional annotation based on sequence comparison alone. Here we carry out sequence analysis and molecular modelling of two families of bacterial dPGM homologues, one the SixA phosphoprotein phosphatases, the other containing various proteins of no known molecular function. The models show how SixA proteins have adapted to phosphoprotein substrate and suggest that the second family may also encode phosphoprotein phosphatases. Unexpected variation in catalytic and substrate-binding residues is observed in the models.

A divergent archaeal member of the alkaline phosphatase binuclear metalloenzyme superfamily has phosphoglycerate mutase activity.

The hyperthermophilic archaeon Methanococcus jannaschii uses several non-canonical enzymes to catalyze conserved reactions in glycolysis and gluconeogenesis. A highly diverged gene from that organism has been proposed to function as a phosphoglycerate mutase. Like the canonical cofactor-independent phosphoglycerate mutase and other members of the binuclear metalloenzyme superfamily, this M. jannaschii protein has conserved nucleophilic serine and metal-binding residues. Yet the substrate-binding residues are not conserved. We show that the genes at M. jannaschii loci MJ0010 and MJ1612 encode thermostable enzymes with phosphoglycerate mutase activity. Phylogenetic analyses suggest that this gene family arose before the divergence of the archaeal lineage.

A superfamily of metalloenzymes unifies phosphopentomutase and cofactor-independent phosphoglycerate mutase with alkaline phosphatases and sulfatases.

Sequence analysis of the probable archaeal phosphoglycerate mutase resulted in the identification of a superfamily of metalloenzymes with similar metal-binding sites and predicted conserved structural fold. This superfamily unites alkaline phosphatase, N-acetylgalactosamine-4-sulfatase, and cerebroside sulfatase, enzymes with known three-dimensional structures, with phosphopentomutase, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, phosphoglycerol transferase, phosphonate monoesterase, streptomycin-6-phosphate phosphatase, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1, and several closely related sulfatases. In addition to the metal-binding motifs, all these enzymes contain a set of conserved amino acid residues that are likely to be required for the enzymatic activity. Mutational changes in the vicinity of these residues in several sulfatases cause mucopolysaccharidosis (Hunter, Maroteaux-Lamy, Morquio, and Sanfilippo syndromes) and metachromatic leucodystrophy.

Muscle phosphoglycerate mutase deficiency.

A 52-year-old man complained since adolescence of cramps and pigmenturia after 15 to 30 minutes of intense exercise. There was no family history of neuromuscular diseases, and strength was normal. The rise of venous lactate after forearm ischemic exercise was abnormally low. Histochemical and ultrastructural studies of a muscle biopsy showed mild increase of glycogen, which was confirmed by biochemical analysis. Studies of anaerobic glycolysis in vitro showed decrease lactate formation with glycogen and with all hexosephosphate glycolytic intermediates, suggesting a defect below the phosphofructokinase reaction. Muscle phosphoglycerate mutase (PGAM) activity was 5.7% of the lowest control, while all other enzymes of glycolysis had normal activities. Electrophoretic, heat lability, and mercury inhibition studies showed that the small residual activity of PGAM in the patient's muscle was represented by the brain (BB) isoenzyme, suggesting a genetic defect of the M subunit that predominates in normal muscle. The prevalence of the BB isoenzyme in other tissues, including muscle culture, may explain why symptoms were confined to muscle.