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SULFUR METABOLISM IN ASPERGILLUS NIDULANS:
PATHWAYS AND REGULATION1
Andrzej Paszewski, Renata Natorff, Marzena Sieńko, Jerzy Brzywczy and Irmina
Lewandowska
Department of Genetics, Institute of Biochemistry and Biophysics PAS, Pawińskiego 5a,
Warszawa, Poland
Abstract
-Figures 2, 3 and 4 are not cited in the text – please cite them in the correct places
-comments to Fig1 and its legend: (1) what is in italics – presumably genes names but explain
that there are genes names in the legend, (2)“cB” should be probably “cysB” etc., (3) show where
is SAH lyase if possible –this would help to explain Fig. 4., (4) replace “—“ with “2-“
-comments to Fig.4 and its legend: perhaps after explaining in Fig.1 where is SAH lyase it would
be possible to have the legend as corrected.
The final product of the sulfate assimilation pathway, sulfide, is incorporated into serine
or homoserine carbon chain, forming cysteine or homocysteine, respectively. There are
three types of pathway organization by which sulfur amino acids are synthesized and
interconverted. Plants, Schizosaccharomyces pombe and Enterobacteriaceae synthesize
de novo cysteine, which is sequentially converted to methionine. Saccharomyces
cerevisiae makes homocysteine, which serves as a precursor of both cysteine and
methionine. Only in Aspergillus nidulans there are alternative pathways for cysteine and
homocysteine de novo synthesis. Impairment of both pathways results in cysteine
auxotrophy. It was found, however, that the route involving homocysteine synthase,
cystathionine β-synthase and cystathionine γ-lyase (Fig. 1, steps 11, 12, 13) functions
efficiently when the main pathway of cysteine synthesis from O-acetylserine is
impaired. In consequence, mutations in the cysA and cysB genes lead to elevated
synthesis of homocysteine by homocysteine synthase so they suppress mutations in the
metB, metG and metA genes. In fact, mutations in cys genes are readily isolated as
suppressors of mutations in the met genes.
Another types of isolated suppressor mutations, exerting the same effect as mutations
in cys genes, are mutations in regulatory genes. Some of them are located in the four
negative-acting scon genes (Natorff et al. 1993), others in the positive-acting metR gene
encoding a sulfate assimilation transcriptional activator, which belongs to the bZIP
family (Natorff et al. 2003).
Scon and metR genes constitute the sulfur metabolite repression (SMR) system,
which controls the expression of genes encoding the sulfate assimilation pathway and
Sulfur Metabolism in Higher Plants, pp. xx-xx
Edited by A. Sirko et al.
 2009 Backhuys Publishers, Leiden, The Netherlands
homocysteine synthase. Two scon genes encode components of a multiprotein ubiquitin
ligase of SCF-type (Skp1-Cullin-Fbox). The A. nidulans SconB protein (Natorff et al.
1998) is an orthologue of S. cerevisiae Met30p that determines specificity of SCF ligase.
It contains an F-box motif that interacts with the Skp1 adaptor protein encoded by A.
nidulans sconC (Piotrowska et al. 2000). The S. cerevisiae orthologue of SconC (Skp1p)
is an essential subunit of SCF complexes. SCF ligases are elements of the ubiquitin
proteasome system that regulates many cellular processes, including nutrient sensing.
Under conditions of cysteine excess, the level or activity of the MetR protein is
negatively controlled by the SCFSconB ligase. Mutations in the scon genes lead to loss of
sulfur metabolite repression. In this way they cause overproduction of sulfur compounds
from sulfate even in the presence of cysteine or methionine, probably as a result of
inefficient degradation of the MetR protein.
Fig. 1. An outline of sulfur containing amino acid metabolism and folate metabolism in
Aspergillus nidulans. Enzymes: 1, sulfate permease; 2 , ATP-sulfurylase; 3, AdoPS-kinase; 4,
PadoPS-reductase: 5, sulfite reductase; 6, serine transacetylase; 7, cysteine synthase (Oacetylserine sulfhydrylase); 8, homoserine transacetylase; 9, cystathionine -synthase; 10,
cystathionine -lyase; 11, homocysteine synthase; 12, cystathionine -synthase; 13, cystathionine
-lyase; 14 , dihydrofolate reductase; 15, serine hydroxymethyltransferase; 16,
methylenetetrahydrofolate reductase; 17, methionine synthase; 18, S-adenosyl-methionine
synthase; 19, S-adenosylhomocysteine hydrolase. AdoMet, S-adenosylmethionine; AdoHcy, Sadenosylhomocysteine; AdoPS, adenosine 5-phosphosulfate; PadoPS, adenosine 3-phosphate 5phosphosulfate;
CH2H4PteGlun, methylenetetrahydrofolylpolyglutamate; CH3H4PteGlun,
5-methyltetrahydrofolylpolyglutamate.
Fig. 2. Mutations in the cysB, sconB, sconC and metR genes suppress metB3 and metA17
mutations. Growth of mutants on minimal medium with (or without) 0,25 mM methionine.
Fig. 3. Ubiquitination of the MetR protein by SCFSconB ligase at high concentration of cysteine - a
low-molecular weight effector of this complex. Ubiquitinated MetR is directed to degradation or
inactivated.
Losses of function mutations in the metR gene are epistatic to mutations in the
negative-acting scon genes and lead to methionine auxotrophy. Dominant-type
mutations in the metR gene (MetRd) lead to suppression of mutations in the metA, metB
and metG genes. In these mutants a conserved N-terminal phenylalanine (F48) of MetR
is changed to serine or leucine. Mutated MetR(F48S) may be more resistant to degradation.
Thus, the efficient synthesis of homocysteine (via homocysteine synthase) can be
achieved by three different classes of mutations: i) Block in cysteine synthesis (lowered
level of regulatory effector), cysA and cysB mutations; ii) SCF complex damage (MetR
protein excess), sconB and sconC mutations; iii) MetR protein stabilization – MetRd
mutations Both scon and metR ortologues are present in all filamentous fungal species
for which genomic sequences are available.
Fig. 4. Homocysteine up-regulates genes that encode enzymes involved in homocysteine
metabolism: A) metH, metA, metF, mecA, mecB, mecC, and SAH lyase encoding gene. B)
folylpolyglutamate synthase (FPGS) and dihydrofolate synthase (DHFS) encoding genes.
Northern analysis of the wild type strain grown in minimal medium suplemented with Lhomocysteine (1, 2 or 3 mM), or with L-methionine (5 mM).
Homocysteine occupies a branch point in methionine, cysteine, and
S-adenosylmethionine metabolism. Elevated levels of homocysteine have various
deleterious effects. Cells maintain a low level of homocysteine mainly by means of two
metabolic pathways. The first is conversion to cysteine, a precursor of glutathione, the
major redox regulating metabolite of the cell. The second is methylation to methionine
catalyzed by methionine synthase in a reaction that requires methyltetrahydrofolate.
About half of homocysteine formed is conserved by remethylation to methionine in the
"methionine cycle". The other half is irreversibly converted to cysteine by cystathionine
-synthase and cystathionine -lyase.
We have shown that homocysteine up-regulates genes encoding enzymes involved in
methionine synthesis: metH coding for methionine synthase (Kacprzak et al. 2003) as
well as metA and metF genes encoding methylenetetrahydrofolate reductase isoenzymes
which synthesize methyltetrahydrofolate (Sieńko et al. 2007). Homocysteine induces
also mecA and mecB genes encoding cystathionine -synthase and cystathionine -lyase
necessary for its conversion to cysteine. Recently we have shown that genes encoding
methionine catabolic enzymes, S-adenosylmethionine synthase (mecC) and
S-adenosylhomocysteine hydrolase, are up-regulated by homocysteine as well as the two
genes encoding folate cycle enzymes: folylpolyglutamate synthase and dihydrofolate
synthase.
Since higher concentrations of homocysteine are toxic to the cell, derepression of
these enzymes by homocysteine is an effective means to remove an excess of this amino
acid. We call the set of genes up-regulated by homocysteine the „homocysteine
regulon”. However, the molecular mechanism of this regulation is not yet elucidated.
References
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