Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter

Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate. The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step. The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis. Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations. Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation. The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations. The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants. This indicates that the effects of the mutations were cumulative. The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase. Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate. Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM.     

Repair in Schizosaccharomyces pombe as measured by recovery from caffeine enhancement of radiation-induced lethality.

Inhibition of DNA repair by caffeine is manifested in Schizosaccharomyces pombe wild-type cells as an enhancement of UV- or gamma-irradiation-induced lethality. The progress of DNA repair processes involving one or more caffeine-sensitive steps may be conveniently followed by measuring the concomitant decrease of this lethal enhancement effect. By measuring, during post-irradiation incubation, the ability of cells to overcome susceptibility to repair inhibition by caffeine, we have determined the time course and requirements for repair in S. pombe. Recovery began immediately and took 150-200 min after gamma-irradiation and more than 500 min after UV-irradiation, for exposures which gave about 10% survival in the absence of caffeine. An incubation medium capable of supporting growth was required for caffeine-sensitive repair; no recovery occurred under liquid holding conditions. Survival curves after various recovery times indicated that a logarithmic phase cell population was homogeneous with respect to caffeine-sensitive repair of both UV- and gamma-ray-induced damage. Recovery from caffeine inhibition was compared for cells of different physiological states (logarithmic and stationary phase); although the importance of the physiological state was not the same for the two types of radiation, recovery was found to occur more rapidly in the more radiation-resistant state, in each case.     

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.     

Regulation of D-glucose-6-phosphate dehydrogenase from Schizosaccharomyces pombe.

D-Glucose-6-phosphate dehydrogenase is a regulatory enzyme of the oxidative pentose phosphate pathway in Schizasaccharomyces pombe. The enzyme is subject to negative cooperative regulation by D-glucose-6-phosphate as characterized by the Hill coefficient of 0.68 +/- 0.04. D-Glyceraldehyde-3-phosphate and D-ribulose-5-phosphate rectify the negative cooperativity as evidenced from a change in the Hill coefficients to 0.98 +/- 0.05 and 1.02 +/- 0.05, respectively. These pentose phosphate pathway intermediates also inhibit the enzyme competitively with respect to D-glucose-6-phosphate. Thus, D-glucose-6-phosphate dehydrogenase provides an avenue for regulating the partitioning of D-glucose between the redundant branches of the oxidative phosphate pathway in S. pombe.     

Specific initiation at an origin of replication from Schizosaccharomyces pombe.

Using a genetic assay for efficient autonomous replication, we have isolated from Schizosaccharomyces pombe a 6.2-kb fragment which shows the properties expected of an origin of DNA replication in S. pombe. A 2.8-kb subclone of the fragment has the same replication properties. Two-dimensional gel analysis of replication intermediates throughout plasmids carrying the 6.2- or 2.8-kb fragments shows that replication initiates only in a specific region, which can be localized to within several hundred base pairs, in the fragments. This region is also a site of replication initiation in the S. pombe chromosome where the fragments normally reside. These results provide strong evidence that initiation of replication in S. pombe is localized and mediated by specific DNA sequence signals.     

Repair in Schizosaccharomyces pombe as measured by recovery from caffeine enhancement of radiation-induced lethality

Molecular Genetics and Genomics - Inhibition of DNA repair by caffeine is manifested in Schizosaccharomyces pombe wild-type cells as an enhancement of UV- or γ-irradiation-induced lethality....     

Spectral characteristics of cadmium-containing phytochelatin complexes isolated from Schizosaccharomyces pombe.

Phytochelatins, heavy-metal-containing peptides with structures (gamma EC)nG, where n = 2-8, have been isolated from higher plants and the fission yeast Schizosaccharomyces pombe. The present work describes the isolation and characterization of several naturally occurring mixed complexes of these peptides from S. pombe exposed to 1 mM CdCl2. A lower-molecular-mass fraction from Sephadex G-50 chromatography yielded three distinct species on further fractionation. HPLC chromatography revealed the presence of peptides with n = 1-4 in varying amounts in these three complexes, referred to as complexes I, II and III. Stoichiometries are proposed for these complexes, based on [Cd], [SH], [S2-] and the amino acid content. Ultraviolet absorption and magnetic circular dichroism spectra of complexes II and III are similar, whereas the CD spectra of these two complexes are strikingly different. Compared to both complexes II and III, the CD bands of complex I are relatively weak. Ultraviolet absorption, CD and magnetic circular dichroism spectra provide a basis for the discussion of structural differences in these complexes.     

Genetic effects of potassium dichromate in Schizosaccharomyces pombe.

In this review we outline the various factors which may contribute to the non-randomness of intragenic mutational spectra and the occurrence of hot spots. These factors include sample size limitation, particularly for sites of low mutability, and possible regions of low recombination potential. In addition, the nature of the gene product places great restraint on the detectability of either frameshift and premature chain-terminating mutations on one hand, or of the majority of missense mutations on the other. The nature of the Genetic Code itself also limits the mutational spectrum in so far as specific base pair substitutions lead only to a limited number of detectable amino acid replacements.Mutational hot spots may be a special example of the influence of neighbouring base pairs in the mutability of any given base pair. This is apparently true for frameshift mutations which tend to occur in runs of repeated base pairs or base pair doublets. Neigbouring base effects could operate not only at the level of initial reactivity with a mutagen, but also subsequently at the levels of DNA repair, recombination or replication. In some cases rare or modified bases may be responsible for neighbour effects. We suggest specific experimental approaches which seem likely to aid in the elucidation of these problems.     

Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe.

The denaturation by guanidinium chloride of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe was studied. The loss in activity broadly parallels the changes in protein structure detected by fluorescence and c.d. Renaturation can be brought about by dilution of the denaturing agent. These processes were compared with those in the enzymes from baker's yeast and rabbit muscle, which are tetrameric and dimeric respectively. The effects of the cofactor 2,3-bisphosphoglycerate on the structure and stability of the S. pombe enzyme were also investigated.     

A homologue of the ras-related CDC42 gene from Schizosaccharomyces pombe.

A cDNA was isolated from the fission yeast, Schizosaccharomyces pombe, using mixed oligodeoxyribonucleotides encoding part of the GTP-binding site of the ras superfamily. The encoded protein is the homologue of the budding yeast CDC42 gene product and the human proteins, CDC42Hs and G25K.     

Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.     

Protein transport in the permeabilized cell of Schizosaccharomyces pombe.

We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae. We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus. Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol. A glycoprotein of S. pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S. pombe system, but not in the S. cerevisiae system. This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S. pombe.     

Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe.

Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.     

The nucleotide sequence of tRNA tyrosine from the fission yeast Schizosaccharomyces pombe.

The sequence of tRNA tyrosine from the fission yeast Schizosaccharomyces pombe is pCUCCUGAUm1 GGUG psi AGDDGGDDAUCACACor (psi) CCGGUG psi Ai6 AACCGGUUGm7 GUm5C GCUAGT psi CGm1 AUUCUGGUCAGGAGACCAOH. This sequence differs in 30 nucleotides from the tRNA-Tyr seqence of the budding yeast Saccharomyces cerevisiae. It has a unique anticodon stem of only four GC base pairs. The normal fifth pair position of nucleotide 28-44 is occupied by a C-U and in 20% of the tRNA-Tyr molecules it is psi-U. This unusual feature and its implications are considered in the discussion.