Andrzej Paszewski

An Aspergillus nidulans mutant lacking cystathionine β-synthase: Identity of L-serine sulfhydrylase with cystathionine β-synthase and its distinctness from O-acetyl-L-seline sulfhydrylase

A monogenic mutant of Aspergillus nidulans simultaneously lacking cystathionine β-synthase (EC 4.2.1.13) and L-serine sulfhydrylase was isolated. The study of this mutant and the wild-type strain indicates that: 1. 1. In A. nidulans, O-acetyl-L-serine sulfhydrylase and L-serine sulfhydrylase are distinct enzymes. 2. 2. L-Serine sulfhydrylase is apparently identical with cystathionine β-synthase. 3. 3. The physiological role of the latter enzyme appears to be in cystathionine synthesis rather than in cysteine synthesis from serine.

Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation.

Using a Saccharomyces cerevisiae strain having the activities of serine O‐acetyl‐transferase (SATase), O‐acetylserine/O‐acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine β‐synthase (β‐CTSase) and cystathionine γ‐lyase (γ‐CTLase), we individually disrupted CYS3(coding for γ‐CTLase) and CYS4 (coding for β‐CTSase). The obtained gene disruptants were cysteine‐dependent and incorporated the radioactivity of 35S‐sulphate into homocysteine but not into cysteine or glutathione. We concluded, therefore, that SATase and OAS/OAH SHLase do not constitute a cysteine biosynthetic pathway and that cysteine is synthesized exclusively through the pathway constituted with β‐CTSase and γ‐CTLase; note that OAS/OAH SHLase supplies homocysteine to this pathway by acting as OAH SHLase. From further investigation upon the cys3‐disruptant, we obtained results consistent with our earlier suggestion that cysteine and OAS play central roles in the regulation of sulphate assimilation. In addition, we found that sulphate transport activity was not induced at all in the cys4‐disruptant, suggesting that CYS4 plays a role in the regulation of sulphate assimilation. Copyright

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapBl), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds. scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes apear to be inducible by homocysteine and repressible by methionine (or Sadenosylmethionine).

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes

The identification, isolation and characterization of a new Aspergillus nidulans positive‐acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative‐acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA‐binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys‐3 gene does not substitute for the metR function, a chimeric metR gene with a cys‐3 bZIP domain is able to transform the ΔmetR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys‐3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP‐sulphurylase, and sulphur controller –sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.

THE ASPERGILLUS NIDULANS SULPHUR REGULATORY GENE SCONB ENCODES A PROTEIN WITH WD40 REPEATS AND AN F-BOX

Abstract The Aspergillus nidulans gene sconB, one of the four identified genes controlling sulphur metabolite repression, was cloned and analysed. It encodes a polypeptide of 678 amino acids containing seven WD repeats characteristic of the large WD40 family of eukaryotic regulatory proteins. The SCONB protein has nuclear localisation signals and is very similar to the Neurospora crassa SCON2 and Saccharomyces cerevisiae Met30 proteins, both of which are involved in the regulation of sulphur metabolism. The N. crassa scon-2 gene complements the sconB2 mutation. All three proteins also contain a newly identified motif, the F-box, found in a number of eukaryotic regulatory proteins. This motif is responsible, at least in some cases, for ubiquitin-mediated proteolysis. The sconB transcript is derepressed under sulphur limitation conditions and partly repressed by high methionine.

Regulation of S-amino acids biosynthesis in Aspergillus nidulans

SummaryIt was found that in Aspergillus nidulans the enzymes of the sulfate assimilation pathway and O-acetylhomoserine sulfhydrylase are under cysteine- and/or homocysteine, but not methionine- or S-adenosylmethionine-mediated regulation. These enzymes are repressed when the cells are grown in the presence of cysteine or homocysteine even in conditions where cysteine cannot be a precursor of homocysteine and vice versa. This was demonstrated by using mutants with impaired cystathionine cleavage enzymes. Thus, these two amino acids can substitute each other as the regulatory effectors. The addition of methionine causes repression only in conditions when it can be metabolized to homocysteine. The mutant cysA1 with a block at the serine transacetylase step is a prototroph owing to the existence of an alternative pathway for cysteine synthesis involving the enzymes: homocysteine synthase, cystathionine β-synthase and γ-cystathionase. All the three enzymes as well as those of the sulfate assimilation pathway are derepressed in this mutant. CysA1 mutation supresses the meth55 mutant blocked at β-cystathionase owing to the derepression of homocysteine synthase, so that the cystathionine cleavage step is bypassed. The results indicate that the pathway involving cystathionine formation is the main one for methionine biosynthesis in A. nidulans. The pathway involving homocysteine synthase is an alternative-conditional one, physiologically effective only when this enzyme is derepressed.

Regulation of sulphate assimilation in Saccharomyces cerevisiae

We examined how the activity of O‐acetylserine and O‐acetylhomoserine sulphydrylase (OAS/OAH) SHLase of Saccharomyces cerevisiae is affected by sulphur source added to the growth medium and genetic background of the strain. In a wild‐type strain, the activity was repressed if methionine, cysteine or glutathione was added to the growth medium. However, in a strain deficient of cystathionine γ‐lyase, cysteine and glutathione were repressive, but methionine was not. In strains deficient of serine O‐acetyltransferase (SATase), OAS/OAH SHLase activity was low regardless of sulphur source and was further lowered by cysteine and glutathione, but not by methionine. From these observations, we concluded that S‐adenosylmethionine should be excluded from being the effector for regulation of OAS/OAH SHLase. Instead, we suspected that S. cerevisiae would have the same regulatory system as Escherichia coli for sulphate assimilation; i.e. cysteine inhibits SATase to lower the cellular concentration of OAS which is required for induction of the sulphate assimilation enzymes including OAS/OAH SHLase. Subsequently, we obtained data supporting this speculation.

Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi

Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine β‐synthase and cystathionine γ‐lyase, two enzymes in the pathway from methionine to cysteine. As a consequence, methionine cannot serve as an efficient sulphur source for the fungus and does not bring about repression of sulphur assimilation, which is under control of the cysteine‐mediated sulphur metabolite repression system. This system operates at the transcriptional level, as was shown for the homocysteine synthase encoding gene. Our results corroborate the growing evidence that cysteine is the major low‐molecular‐weight effector in the regulation of sulphur metabolism in bacteria, fungi and plants. The Sz. pombe homocysteine synthase gene sequence was submitted to GenBank under Accession No. AF012876. Copyright

Gene conversion: observations on the DNA hybrid models

Some features of gene conversion in fungi and their bearing on the hybrid DNA models are discussed. Available experimental data from tetrad analysis seem to give a more complex picture of polarity in intra-genic recombination and of the relations between conversion and post-meiotic segregation, and between conversion and crossing-over, than predicted by the models. A new hypothesis of the mechanism of gene conversion with special attention given to the aspect of asymmetry in this phenomenon is proposed as an alternative to the mechanism suggested by the DNA hybrid models.

Transcriptional regulation of methionine synthase by homocysteine and choline in Aspergillus nidulans.

Roles played by homocysteine and choline in the regulation of MS (methionine synthase) have been examined in fungi. The Aspergillus nidulans metH gene encoding MS was cloned and characterized. Its transcription was not regulated by methionine, but was enhanced by homocysteine and repressed by choline and betaine. MS activity levels were regulated in a similar way. The repression by betaine was due to its metabolic conversion to choline, which was found to be very efficient in A. nidulans. Betaine and choline supplementation stimulated growth of leaky metH mutants apparently by decreasing the demand for methyl groups and thus saving methionine and S -adenosylmethionine. We have also found that homocysteine stimulates transcription of MS-encoding genes in Saccharomyces cerevisiae and Schizosaccharomyces pombe.