Cation-induced thermostability of yeast and Escherichia coli pyrophosphatases

Inorganic pyrophosphatases (PPiases) from both yeast and Escherichia coli were found to be stable against heat denaturation in the presence of Mg2+, as previously observed with the enzymes from thermophilic bacteria. No loss of activity was observed after 1 h of incubation at 50 degrees C and pHs between 6 and 9 in the yeast enzyme, and at 60 degrees C and pHs between 7.2 and 9.2 in the E. coli enzyme. Such an induced thermostability of the E. coli enzyme was detected when Mn2+, Co2+, Ca2+, Cd2+, and Zn2+ were added in place of Mg2+. On the other hand, the degree of induced thermostability of the yeast enzyme was dependent upon the divalent cations used, and Ni2+ and Cu2+ accelerated the heat inactivation. On adding the divalent cations, the difference spectra of the E. coli enzyme always showed negative peaks in the ultraviolet region, but those of the yeast enzyme changed again depending upon the divalent cations. The circular dichroism spectra in the near ultraviolet region of both enzymes greatly differed from each other, but both were not affected so much by adding the divalent cations unlike the thermophilic enzymes from Bacillus stearothermophilus and thermophilic bacterium PS-3. Yeast and E. coli PPiases did not cross-link with the anti-immunoglobulin G's from the thermophilic enzymes, but the thermophilic enzymes did with each other's antisera. The results in the present study indicated that the conformation of PPiase, in which the aromatic amino acid residues were buried in the interior of the protein molecule, was very important for the thermostability and also that the protein structures of PPiases from B. stearothermophilus and thermophilic bacterium PS-3 were very similar to each other, but were very different from those of the mesophilic enzymes.     

Computer modeling of two inorganic pyrophosphatases.

The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related. One, PPA1, is a cytoplasmic enzyme. The other, PPA2, is located in the mitochondria and appears to be energy-linked. The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50%. All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2. The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1. Two cysteines in PPA2 and one in the S. pombe enzyme are located at the catalytic cleft. Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme.     

Immunological cross-reactivity between proton-pumping inorganic pyrophosphatases of widely phylogenic separated species.

Immunological cross-reactivity among three types of inorganic pyrophosphatases, that is, the proton pumping inorganic pyrophosphate synthase (H(+)-PPi synthase) and the soluble inorganic pyrophosphatase, both from Rhodospirillum rubrum, and the vacuolar membrane inorganic pyrophosphatase (H(+)-PPase) from mung bean (Vigna radiata), were examined by means of immunoblot analyses. Antibodies raised against the mung bean H(+)-PPase cross-reacted with the H(+)-PPi synthase from R. rubrum but not with the soluble PPase from R. rubrum. N,N'-dicyclohexylcarbodiimide (DCCD), which inhibits both synthesis and hydrolysis of PPi catalysed by purified and chromatophore H(+)-PPi synthase, binds to the enzyme as shown by fluorography of [14C]DCCD labelling. These results suggest that the R. rubrum H(+)-PPase share close structural similarities with the vacuolar H(+)-PPase from Mung bean.     

Functionally important lysine residues in inorganic pyrophosphatase from E. coli. I. Interaction of inorganic pyrophosphatase with pyridoxal-5'-phosphate

Interaction of inorganic pyrophosphatase from E. coli with pyridoxal-5'-phosphate includes binding of the reagent at the active site through the phosphate group and then a reversible modification of one lysine residue in each of the enzyme's subunit. In the equilibrium state the protein's molecules contain both inactive modified and native subunits. A stable secondary amine is formed upon the sodium borohydride reduction of the modified protein.     

Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria

Soluble inorganic pyrophosphatases (inorganic diphosphatases, EC 3.6.1.1) were isolated and characterized from three phylogenetically diverse cyanobacteria--Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Pseudanabaena sp. PCC 6903--and one anoxygenic photosynthetic bacterium, Rhodopseudomonas viridis (purple nonsulfur). These enzymes were found to be family I soluble inorganic pyrophosphatases with c. 20 kDa subunits with diverse oligomeric structures. The corresponding ppa genes were cloned and functionally validated by heterologous expression. Cyanobacterial family I soluble inorganic pyrophosphatases were strictly Mg(2+)-dependent enzymes. However, diverse cation cofactor dependence was observed for enzymes from other groups of photosynthetic bacteria. Immunochemical studies with antibodies to cyanobacterial soluble inorganic pyrophosphatases showed crossreaction with orthologs of other main groups of phototrophic prokaryotes and suggested a close relationship with the enzyme of heliobacteria, the nearest photosynthetic relatives of cyanobacteria. A slow-growing Escherichia coli JP5 mutant strain, containing a very low level of soluble inorganic pyrophosphatase activity, was functionally complemented up to wild-type growth rates with ppa genes from diverse photosynthetic prokaryotes expressed under their own promoters. Overall, these results suggest that the bacterial family I soluble inorganic pyrophosphatases described here have retained functional similarities despite their genealogies and their adaptations to diverse metabolic scenarios.     

Functionally important lysine residues in inorganic pyrophosphatase from E. coli. II. Isolation and characteristics of modified tryptic peptide and analysis of the functional role of lysine residues controlling enzyme activity

Inactivation of inorganic pyrophosphatase from E. coli by pyridoxal-5'-phosphate is due to the modification of a lysine residue located in the tryptic peptide with the Asp-Leu-Pro-Glu N-terminal sequence. In course of the enzymatic process this lysine-residue appears to be in the protonated state and either operators as the proton donor for the product of the enzymatic reaction or is involved in stabilization of the transition state.     

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.     

Deletions within E. coli plasmids carrying yeast rDNA.

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.     

Active site interactions in oligomeric structures of inorganic pyrophosphatases

Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers.     

Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

A novel subfamily of monomeric inorganic pyrophosphatases in photosynthetic eukaryotes

Two sPPases (soluble inorganic pyrophosphatases, EC 3.6.1.1) have been isolated from the microalga Chlamydomonas reinhardtii. Both are monomeric proteins of organellar localization, the chloroplastic sPPase I [Cr (Ch. reinhardtii)-sPPase I, 30 kDa] is a major isoform and slightly larger protein than the mitochondrial sPPase II (Cr-sPPase II, 24 kDa). They are members of sPPase family I and are encoded by two different cDNAs, as demonstrated by peptide mass fingerprint analysis. Molecular phylogenetic analyses indicated that Cr-sPPase I is closely related to other eukaryotic sPPases, whereas Cr-sPPase II resembles its prokaryotic counterparts. Chloroplastic sPPase I may have replaced a cyanobacterial ancestor very early during plastid evolution. Cr-sPPase II orthologues are found in members of the green photosynthetic lineage, but not in animals or fungi. These two sPPases from photosynthetic eukaryotes are novel monomeric family I sPPases with different molecular phylogenies and cellular localizations.     

E. coli dihydroorotate dehydrogenase reveals structural and functional distinctions between different classes of dihydroorotate dehydrogenases

The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.     

Targeting of E. coli beta-galactosidase to the nucleus in yeast

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.