Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.     

A marsupial phosphoglycerate kinase (PGK) processed pseudogene

A clone that cross-hybridized with a full-length human cDNA PGK probe was isolated from a hill kangaroo (Macropus robustus: Marsupialia) lambda EMBL4 EcoRI genomic library. The clone was sequenced and demonstrated to be a pseudogene, with two deletions (one of 3 bases, the other 24 bases long), one single base insertion, and a nonsense mutation with respect to the functional human X-linked gene. It is flanked by terminal repeats in the 5' and 3' noncoding regions, but it has no 3' poly(A) remnant. The 3' untranslated region has a 34-bp sequence, with 29 bp homologous to the human 3' untranslated region. The overall percentage homology with the mouse and human X-linked PGK indicates that this pseudogene is probably more closely related to eutherian X-linked PGK genes than to the autosomal form. The results also suggest that pseudogenes are of considerable antiquity (greater than 100 MYr) in the mammalian lineage.     

The phosphoglycerate kinases from Trypanosoma brucei. A comparison of the glycosomal and the cytosolic isoenzymes and their sensitivity towards suramin

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2',3'-ATP-Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga. Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308----His and Ala 306----Glu in the adenine-binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogues, but probably prevent phosphoryl-group transfer. Both isoenzymes displayed an activity optimum at pH 6.0-9.0 similar to that for the enzyme of yeast. Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Ki app. = 8.0 microM) to Suramin than either the cPGK (Ki app. = 20 microM) or the enzymes from rabbit muscle (Ki app. = 55 microM), yeast (Ki app. = 167 microM) or S. platensis (Ki app. = 250 microM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.     

Crystal structures of putative phosphoglycerate kinases from B. anthracis and C. jejuni

Phosphoglycerate kinase (PGK) is indispensable during glycolysis for anaerobic glucose degradation and energy generation. Here we present comprehensive structure analysis of two putative PGKs from Bacillus anthracis str. Sterne and Campylobacter jejuni in the context of their structural homologs. They are the first PGKs from pathogenic bacteria reported in the Protein Data Bank. The crystal structure of PGK from Bacillus anthracis str. Sterne (BaPGK) has been determined at 1.68 ? while the structure of PGK from Campylobacter jejuni (CjPGK) has been determined at 2.14 ? resolution. The proteins' monomers are composed of two domains, each containing a Rossmann fold, hinged together by a helix which can be used to adjust the relative position between two domains. It is also shown that apo-forms of both BaPGK and CjPGK adopt open conformations as compared to the substrate and ATP bound forms of PGK from other species.     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

Correlation between conformational stability of the ternary enzyme-substrate complex and domain closure of 3-phosphoglycerate kinase

3-phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N- or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and side-chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain-domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate-induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented [Sinev MA, Razgulyaev OI, Vas M, Timchenko AA & Ptitsyn OB (1989) Eur J Biochem180, 61-66]. Thus, conformational stability correlates with domain closure that requires simultaneous binding of both substrates.     

Mechanisms of Protein Sequence Divergence and Incompatibility

Alignments of orthologous protein sequences convey a complex picture. Some positions are utterly conserved whilst others have diverged to variable degrees. Amongst the latter, many are non-exchangeable between extant sequences. How do functionally critical and highly conserved residues diverge? Why and how did these exchanges become incompatible within contemporary sequences? Our model is phosphoglycerate kinase (PGK), where lysine 219 is an essential active-site residue completely conserved throughout Eukaryota and Bacteria, and serine is found only in archaeal PGKs. Contemporary sequences tested exhibited complete loss of function upon exchanges at 219. However, a directed evolution experiment revealed that two mutations were sufficient for human PGK to become functional with serine at position 219. These two mutations made position 219 permissive not only for serine and lysine, but also to a range of other amino acids seen in archaeal PGKs. The identified trajectories that enabled exchanges at 219 show marked sign epistasis - a relatively small loss of function with respect to one amino acid (lysine) versus a large gain with another (serine, and other amino acids). Our findings support the view that, as theoretically described, the trajectories underlining the divergence of critical positions are dominated by sign epistatic interactions. Such trajectories are an outcome of rare mutational combinations. Nonetheless, as suggested by the laboratory enabled K219S exchange, given enough time and variability in selection levels, even utterly conserved and functionally essential residues may change.     

Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae.

The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -63 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.     

Phosphoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex.

Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.     

The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension.

To determine how microbody proteins enter microbodies, we have previously compared the genes for the cytosolic and glycosomal (microbody) phosphoglycerate kinases (PGKs) of Trypanosoma brucei and found the microbody enzyme to differ from other PGKs and the cytosolic form in two respects: a high net positive charge and a C-terminal extension of 20 amino acids (Osinga et al., 1985). Here we present the comparison of the genes for the cytosolic and glycosomal PGKs of Crithidia fasciculata, another kinetoplastid organism. The amino acid sequences of the two Crithidia isoenzymes are virtually identical, except for a C-terminal extension of 38 amino acids. We conclude that this extension must direct the glycosomal PGK to the glycosome. The extensions of the Crithidia and Trypanosoma enzymes are both rich in small hydrophobic and hydroxyl amino acids.     

Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Comparison of proteolytic susceptibility in phosphoglycerate kinases from yeast and E. coli: modulation of conformational ensembles without altering structure or stability

Escherichia coli phosphoglycerate kinase (PGK) is resistant to proteolytic cleavage while the yeast homolog from Saccharomyces cerevisiae is not. We have explored the biophysical basis of this surprising difference. The sequences of these homologs are 39% identical and 56% similar. Determination of the crystal structure for the E. coli protein and comparison to the previously solved yeast structure reveals that the two proteins have extremely similar tertiary structures, and their global stabilities determined by equilibrium denaturation are also very similar. The extrapolated unfolding rate of E. coli PGK is, however, 10(5) slower than that of the yeast homolog. This surprisingly large difference in unfolding rates appears to arise from a divergence in the extent of cooperativity between the two structural domains (the N and C-domains) that make up these kinases. This is supported by: (1) the C-domain of E. coli PGK cannot be expressed or fold independently of the N-domain, while both domains of the yeast protein fold in isolation into stable structures and (2) the energetics and kinetics of the proteolytically sensitive state of E. coli PGK match those for global unfolding. This suggests that proteolysis occurs from the globally unfolded state of E. coli PGK, while the characteristics defining the yeast homolog suggest that proteolysis occurs upon unfolding of only the C-domain, with the N-domain remaining folded and consequently resistant to cleavage.