Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t 0.5∼10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K 0.5∼100 μM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine.


Introduction
The polyamines spermidine and spermine are tri-and tetravalent organic cations that are essential for the normal growth of eukaryotic organisms (Tabor and Tabor 1984;Pegg 1986).The divalent polyamine putrescine, a product of the ornithine decarboxylase (ODC) reaction, is converted to spermidine and then spermine by successive aminopropyl transfer reactions.The S-adenosylmethionine decarboxylase (AdoMetDC) reaction produces decarboxylated S-adenosylmethionine, which serves as the aminopropyl donor in putrescine and spermidine aminopropyltransferase reactions (catalyzed by spermidine synthase and spermine synthase, respectively).Studies with polyamine auxotrophs of the fungi Saccharomyces cerevisiae and Neurospora crassa have demonstrated that spermidine, the major polyamine in these organisms, serves functions for which putrescine cannot substitute (Cohn et al. 1978;Whitney and Morris 1978;Pitkin and Davis 1990;Balasundaram et al. 1991).In animal cells, both spermidine and spermine are predominant (Watanabe et al. 1991), and inhibitors that block the formation of these polyamines also restrict the growth of cultured cells (Pegg et al. 1988;Kramer et al. 1989).
In eukaryotic cells, AdoMetDC is a (ab) 2 dimer, each half being composed of two non-identical subunits derived by cleavage of a proenzyme precursor.The cleavage generates a pyruvate prosthetic group at the Nterminus of the a subunit, the larger, C-terminal fragment.AdoMetDC belongs to a class of enzymes in which the presence of this pyruvoyl group is essential for activity (van Poelje and Snell 1990).Studies with the yeast and human proenzymes have shown that the cleavage reaction occurs at a glutamyl-serine bond in the sequence YVLSESS (Stanley et al. 1989;Kashiwagi et al. 1990), with the pyruvoyl moiety being derived from the serine.
The activity of AdoMetDC from many eukaryotes is stimulated by putrescine (Pegg et al. 1998).In addition, the processing of the human AdoMetDC proenzyme is also stimulated by putrescine (Kameji and Pegg 1987).These regulatory mechanisms make the synthesis of one substrate of spermidine synthase, decarboxylated Sadenosylmethionine, dependent on the presence of the other, putrescine.Because S-adenosylmethionine serves as a methyl donor, and decarboxylated S-adenosylmethionine cannot ful®ll this role, the regulation of Ado-MetDC activity by putrescine levels prevents the inappropriate decarboxylation of S-adenosylmethionine.By contrast, the enzyme activity of the AdoMetDC of Escherichia coli, in which putrescine is the predominant polyamine, is stimulated by Mg 2+ (Wickner et al. 1970).
In N. crassa, as in other eukaryotes, AdoMetDC activity is regulated by cellular polyamine levels (Pitkin and Davis 1990).In this paper we report the characterization of the spe-2 gene of N. crassa, which encodes an unusually large AdoMetDC, so far peculiar to ®lamentous fungi.We show that changes in AdoMetDC activity in response to polyamines are correlated with similar changes in the abundance of spe-2 mRNA.Our results demonstrate that putrescine is absolutely required for the catalytic activity of N. crassa AdoMetDC.However, putrescine does not stimulate proenzyme processing in N. crassa, even though the amino acid residues required for putrescine stimulation of processing in the human gene product are conserved in the fungal protein.
For RFLP mapping, a set of standard strains (Metzenberg et al. 1984) was obtained from the Fungal Genetics Stock Center at the Department of Microbiology, University of Kansas Medical Center, Kansas City, Kan. (FGSC strains 4411±4430).
Growth and maintenance of N. crassa strains followed standard methods (Davis and de Serres 1970).Cultures were supplemented with 1 mM arginine hydrochloride, 1 mM putrescine dihydrochloride, and 1 mM spermidine trihydrochloride, where indicated.
Plasmids, cosmid DNA preparation, and N. crassa transformation The Orbach/Sachs pMOcosX genomic library (Orbach and Sachs 1991) was obtained from the Fungal Genetics Stock Center as an ordered set of 50 96-well microtiter plates containing a total of 4800 individual clones.For screening by complementation, pools of 96 cosmid clones were replicated onto LB agar containing ampicillin (100 lg/ml) and grown overnight at 37 °C.Bacteria were harvested as a pool from the agar plates and cosmid DNA was isolated using a Qiagen plasmid midi kit.Cosmid DNA (5 lg) was used for transformation of N. crassa spheroplasts (Orbach et al. 1986).Cosmid pools that complemented strain IC2798-15 (spe-1, spe-2, aga) were selected by their ability to enable the recipient to use putrescine as a polyamine precursor.A single complementing plasmid was identi®ed in an active pool, using a sib selection strategy (Vollmer and Yanofsky 1986).
Restriction fragments of N. crassa genomic DNA from the complementing cosmid were subcloned into pSP72 vectors (Promega): pWVC1 contains a 6-kb EcoRI fragment with the spe-2 gene at the 5¢ end; pWVC2 contains the same insert as pWVC1, in the opposite orientation; pSPE2 contains a EcoRI-SmaI fragment of pWVC2, in which the spe-2 gene is located; and pSP6-SPE2 contains a HincII-SmaI fragment carrying the spe-2 gene with the AdoMetDC start codon downstream of the SP6 promoter.The pSP6-SPE2 plasmid was constructed by digesting pWVC2 with HindIII at a unique restriction site, in the pSP72 multiple cloning site, between the SP6 promoter and the EcoRI insert.This site was ®lled in, to generate blunt ends, using Klenow DNA polymerase (Sambrook et al. 1989), and the linear plasmid was subsequently cut at a unique BamHI site in the spe-2 coding region.A HincII-BamHI fragment containing the 5¢ end of the spe-2 coding region was directionally cloned into the HindIII + BamHI-cleaved pWVC2 plasmid.Blunt-end ligation joined the ®lled HindIII site in the multiple cloning site to the HincII site found 26 bp upstream of the initiating ATG of the spe-2 coding region.
Nucleic acid isolation and analysis N. crassa genomic DNA was isolated from lyophilized mycelial powders (Bainbridge et al. 1990).Small-scale plasmid DNA preparations were obtained by the alkaline lysis method as previously described (Williams et al. 1992).Plasmid DNA for sequencing was prepared using a Qiagen plasmid midi kit, following the manufacturer's protocol.Plasmids for sequencing were generated using convenient restriction sites in pSPE2 to generate overlapping DNA fragments that were then subcloned into pSP72 vectors.DNA sequencing was carried out using SP6 and T7 primers ¯anking the pSP72 multiple cloning site.DNA sequencing was performed by the University of Chicago Cancer Research Center's DNA sequencing facility (http://cancer-seqbase.uchicago.edu).Chromatograms were manually inspected and edited using Chromas software (version 1.44; http://www.technelysium.com.au/chromas.html),and sequences were assembled using the SeqMan program in the DNASTAR program suite.Sequence information was analyzed using the DNASTAR program suite and the BLAST sequence similarity search tool at the National Center for Biotechnology Information, Bethesda, Md.(http://www.ncbi.nlm.nih.gov/BLAST/).
N. crassa RNA was prepared as previously described (Williams et al. 1992), with minor modi®cations.Wet mycelial pads were collected by ®ltration, frozen, lyophilized overnight, and powdered by vortexing in a tube with a spatula.Dissolution of RNA was routinely followed by a brief (2 min) centrifugation (10,000 ´g) to remove insoluble material.

Restriction fragment length polymorphism mapping
In RFLP mapping of the spe-2 cosmid in the N. crassa genome (Metzenberg et al. 1984), random-primer labeling (Sambrook et al. 1989) of a NotI insert from the X2:B09 cosmid was used to generate probes.This probe recognized an EcoRI polymorphism between the Oak Ridge and Mauriceville strains.Southern analysis (Sambrook et al. 1989) of EcoRI-digested genomic DNA from these strains and a standard set of 18 of their progeny was carried out using the X2:B09 insert as a probe.

Extraction and assay of AdoMetDC activity
Cell extracts were prepared as described previously (Pitkin and Davis 1990).Brie¯y, 250±1000 ml exponential cultures of strain IC3 were harvested by ®ltration, and extraction was carried out at 4 °C.Wet pads were ground with sand in extraction buer (50 mM potassium phosphate pH 7.3, 2 mM dithiothreitol, 1 mM MgCl 2 , 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl ¯uoride).The crude extract was subjected to centrifugation (12,500 ´g, 15 min) and the supernatant was desalted using Sephadex G-25, which removed endogenous polyamines.Crude extracts were immediately used for AdoMetDC assays (Pitkin and Davis 1990).

In vitro synthesis and processing of AdoMetDC proenzyme
The pSP6-SPE2 plasmid, in which the coding region for the N. crassa AdoMetDC proenzyme lies downstream from the SP6 promoter, was used in transcription and translation reactions with the TNT SP6 Coupled Reticulocyte Lysate system (Promega) to measure AdoMetDC processing by the method of Xiong et al. (1997), who established that this preparation contains less than 1 nM putrescine.Reactions were carried out following the manufacturer's protocol, with the modi®cations indicated below.A 50-ll reaction contained 50% TNT rabbit reticulocyte lysate, 0.5 lg of pSP6-SPE2 plasmid DNA, 1´TNT reaction buer, 20 pmol of [ 35 S]methionine (1000 Ci/mmol), the other 19 amino acids at 20 lM each, 40 units of RNasin, and 1 ll of TNT SP6 RNA polymerase.In reactions containing putrescine, the polyamine was added to a ®nal concentration of 1 mM.Reactions were incubated for the indicated periods at 30 °C, and further incorporation of [ 35 S]methionine was blocked by the addition of cycloheximide to a ®nal concentration of 200 lM.Incubation was then continued at 30 °C, and 5-ll samples were removed to 20 ll of sample buer (Gar®n 1990) and heated to 100 °C for 2 min at various times after translation was stopped.Labeled products of the translation reactions were separated by SDS-PAGE on 12% gels, ®xed, dried, and exposed to autoradiographic ®lm.

Identi®cation and mapping of a cosmid containing the spe-2 gene
To clone the spe-2 gene, we screened the Orbach/Sachs pMOcosX genomic library by sib-selection (Staben et al. 1989) for complementation of a strain carrying the spe-2 mutation.Strains containing the spe-2 JP100 allele are unable to synthesize spermidine from putrescine; however, the spermidine pool present in spe-2 mutant cells at the time of inoculation is sucient to allow growth for several doubling times before this polyamine pool is exhausted and growth stops (Pitkin and Davis 1990).This ``leaky'' growth of the spe-2 mutant was eliminated by using a spe-1, spe-2 double mutant (strain IC2798-15) lacking both ODC and AdoMetDC activities.The double mutant failed to grow in putrescine-supplemented medium, con®rming its speci®c and absolute requirement for spermidine.The strain was used as a transformation recipient to screen the N. crassa genomic library for a cosmid that would enable this strain to grow on putrescine.A single complementing cosmid, X2:B09, was identi®ed.
An approximately 30-kb NotI fragment of the X2:B09 cosmid was used to generate a random-primer labeled probe for RFLP mapping.An EcoRI polymorphism between the Oak Ridge (O) and Mauriceville (M) parental strains was detected, and recombinants from a cross between these strains were scored for this dierence (Fig. 1).Comparison of the mapping data with the available RFLP maps (Nelson et al. 1998) indicates that the genomic DNA in cosmid X2:B09 segregates with markers for the loci spe-1, am and inl.The spe-2 locus is located near (20 cM) the inl locus on the right arm of Linkage Group V, 6 and 7 cM to the left of the am and spe-1 loci, respectively (Pitkin and Davis 1990) (Fig. 1).The ability of cosmid X2:B09 to complement the spe-2 mutation and its map localization in the genome at the same position as the spe-2 locus strongly suggests that the cosmid contains the spe-2 gene.
The spe-2 gene encodes the S-adenosylmethionine decarboxylase proenzyme A mutation at the spe-2 locus eliminates AdoMetDC activity, and AdoMetDC activity is kinetically altered in revertants of this mutant (Pitkin and Davis 1990).This suggests that the spe-2 locus is the structural gene for AdoMetDC.To identify a DNA fragment from cosmid X2:B09 containing the spe-2 gene, we ®rst tested various restriction endonucleases for their ability to destroy the complementation activity of the cosmid.Among the enzymes tested, BamHI, ClaI, KpnI, and XhoI eliminated the ability of the cosmid DNA to complement the spe-2 mutation, suggesting that sites for these enzymes lay within the spe-2 gene.Subcloning was carried out using DNA fragments generated from the X2:B09 cosmid by digestion with EcoRI, an enzyme that did not aect complementation activity.For sequence analysis, we subcloned a 3.6-kbEcoR I-Sma I fragment, which complemented the spe-2 mutation, into plasmid pSPE2 (Fig. 2) and sequenced the insert.
The sequence of this 3663-bp DNA fragment (Gen-Bank Accession No. AF151380) revealed a continuous ORF of 1512 nucleotides, encoding a 503 amino acid polypeptide with a predicted molecular weight of 54,721 Da.An examination of public databases using the BLAST search algorithm (Altschul et al. 1997) revealed that this sequence represented a reading frame with homology to the AdoMetDC proenzyme from numerous eukaryotic sources.In agreement with the results of the complementation studies, restriction sites for the endonucleases that eliminated complementing activity of the spe-2 cosmid lay within the predicted AdoMetDC open reading frame (Fig. 2).These results, and those of the RFLP mapping studies, indicate that spe-2 is the N. crassa structural gene for AdoMetDC.
The context of the initiator methionine codon in the putative spe-2 ORF, GACTCAAAATGTCT, is similar to the consensus for sequences surrounding initiation codons of known coding regions in N. crassa, CNNNCA(A/C)(A/C)ATGGCT (Edelmann and Staben 1994).In addition, the presence of an in-frame stop codon, 66 nucleotides upstream of this methionine codon, and the relative position of the predicted N-terminus compared to other eukaryotic AdoMetDC sequences (see below) supports the assumption that this is the translational start codon.The coding region shows a bias for codons with C or G in the third position, typical of N. crassa coding regions, and this coding region is terminated by a TAA codon, the most frequently used stop codon in N. crassa (Edelmann and Staben 1994).
The deduced amino acid sequence of the N. crassa AdoMetDC proenzyme shows moderate homology to those of yeast (S. cerevisiae, 30% identity), and mammals (human, 28% identity), and less to that of plants (potato, 23% identity).Although overall identity is low when these AdoMetDC proenzymes are compared to that of N. crassa, sequence identity is highly conserved in several regions (Fig. 3).These regions include residues involved in proenzyme processing, enzyme activity, and putrescine stimulation of these processes in other eukaryotes.The N. crassa proenzyme contains the sequence YLLSESSMFV (aa 98±107), which is nearly identical to the sequence surrounding the site of proenzyme cleavage in AdoMetDC proenzymes from human (YVLSESSMFV; Stanley et al. 1989) and yeast (YVL-SESSLFI; Kashiwagi et al. 1990).The amino acid residues corresponding to those of the human proenzyme previously shown to be required for catalytic activity (Glu8, Glu11, Ser68, Cys82, Ser229, and His243 in the human sequence), and putrescine stimulation of processing and activity (Glu11, Glu15, Arg76, Lys80, Asp174, Glu178, and Glu256) are identical or similar in the N. crassa AdoMetDC (Fig. 3) (Stanley and Pegg 1991;Stanley et al. 1994;Xiong et al. 1997Xiong et al. , 1999) ) .The conservation of these residues suggests that proenzyme processing and the catalytic activity of the N. crassa enzyme might be stimulated by putrescine.
The deduced length of the N. crassa AdoMetDC proenzyme (503 amino acids) makes it the largest iden-ti®ed thus far ± more than 100 residues longer than the S. cerevisiae proenzyme (396 amino acids).Two large segments in the N. crassa sequence (Ser216 to Pro244, and Ala281 to Cys306) account for most of the additional amino acids when compared to the S. cerevisiae proenzyme.In addition, the AdoMetDC proenzymes from S. cerevisiae and N. crassa contain N-terminal extensions when compared to those of mammalian or plant origin.
Examination of N. crassa expressed sequence tag (EST) databases at the University of New Mexico and nidulans.The similarity of the two enzymes with respect to the presence of additional amino acids suggests that the N. crassa sequence lacks introns in these positions, and that the unusually long proenzyme sequence may be characteristic of ®lamentous fungi.

Characterization of the spe-2 transcript
Because the predicted coding region of the spe-2 gene contains short amino acid sequences homologous to those of other eukaryotic AdoMetDC proenzymes throughout its entire length, and lacks signals normally associated with intron splicing in N. crassa genes, we assumed the coding region lacked intronic sequences.To con®rm the absence of introns, primers ¯anking the spe-2 coding region were used to amplify this region from the spe-2 mRNA by RT-PCR, and from the cloned gene by conventional PCR.The ampli®ed products were then subjected to restriction analysis with BamHI and compared (Fig. 5).The results show that PCR ampli®cation from spe-2 cDNA and genomic DNA results in products of identical size.Digestion of the ampli®ed genomic DNA with BamHI generates fragments of 740 and 820 bp in length.Because the average N. crassa intron is approximately 70 bases in length (Edelmann and Staben 1994), dierences in the size of the ampli®ed cDNA due to the presence of introns would have been detectable.We conclude that the spe-2 coding region lacks introns, and this is supported further by the similarity of the large carboxyl portion of the N. crassa coding region to the predicted sequence encoded by the A. nidulans cDNA.
Northern analysis (Fig. 6) indicates that spe-2 mRNA is expressed as a single 2.0-kb transcript.Cells grown in minimal or spermidine-supplemented medium have little spe-2 mRNA.Starvation for polyamines, brought about by growth of an aga (arginaseless) Fig. 3 Sequence alignment of AdoMetDC proenzyme sequences from human, yeast, potato and N. crassa.The amino acid sequences of AdoMetDC proenzymes from human (Hs, GenBank M21154), N. crassa (Nc), Saccharomyces cerevisiae (Sc, GenBank M38434), and potato (So, GenBank S74514) were aligned by the CLUSTAL method.Residues that are identical in N. crassa and at least one other proenzyme are shaded.The conserved proenzyme cleavage site in human, yeast, and potato is indicated in bold letters.Additional residues in the human sequence ± those in the active-site pocket and those essential for catalytic activity (Glu8, Glu11, Ser68, Cys82, Ser229, and His243) are indicated by the asterisks.Together with Glu11, four additional residues required for putrescine stimulation of human proenzyme processing (Lys80, Asp174, Glu178, and Glu256) are indicated by plus signs.(see Ekstrom et al. 1999 for details) mutant on arginine, or growth of a spe-1 mutant in the absence of polyamine supplementation, leads to a 2-to 4-fold increase in the abundance of spe-2 mRNA, similar to the changes in N. crassa AdoMetDC activity detected under the same conditions (Pitkin and Davis 1990).Depletion of cellular polyamines also causes an increase in the abundance of ODC mRNA, encoded by the spe-1 gene of N. crassa (Williams et al. 1992;Pitkin et al. 1994).Inspection of the regions of the spe-2 and spe-1 genes upstream of their coding regions, however, fails to reveal signi®cant homology.
An ORF capable of encoding a 27-residue peptide lies 509 nucleotides upstream of the AdoMetDC coding region.Because upstream ORFs (uORFs) are regulatory features of mammalian AdoMetDC genes (Ruan et al. 1996), we wished to determine whether this potential uORF resided within the spe-2 transcribed region.Northern analysis was carried out using a SacI-EcoRV probe that overlaps the uORF.This probe failed to detect any transcript, indicating that this upstream region was not part of the spe-2 mRNA.

Stimulation of N. crassa AdoMetDC activity by putrescine
The activity of AdoMetDC from the fungi S. cerevisiae and A. nidulans (Poso et al. 1975;Stevens et al. 1976), the trypanosomes Trypanosoma brucei and T. cruzi (Tekwani et al. 1992;Persson et al. 1998), the nema-Fig.4 Comparison of the predicted amino acid sequences of AdoMetDC from N. crassa and an A. nidulans cDNA.The CLUSTAL method was used to align amino acids 199±503 of the predicted N. crassa AdoMetDC proenzyme and a conceptual translation of an ORF from the partial A. nidulans cDNA m0f09a1 (GenBank Accession No. AF153765).The predicted partial amino acid sequence of the A. nidulans proenzyme is numbered from the most N-terminal residue encoded by the cDNA.Amino acids which are identical in both predicted coding sequences are highlighted.The two sequence insertions found in N. crassa that are absent in most other eukaryotes are overlined Fig. 5 Con®rmation of the absence of introns from the coding region of the spe-2 gene.Reverse transcription of total N. crassa DNA was carried out using primer P2 (see Materials and Methods), followed by PCR ampli®cation of the spe-2 coding region using primers P1 and P2.No product appeared if reverse transcriptase was omitted in the ®rst step (not shown).As a control, the PCR product of the spe-2 sequence in pSPE2 was also made.The products were digested with BamH1 and compared after agarose gel electrophoresis.Lane 1, PCR product of total RNA after reverse transcription; lane 2, PCR product of the plasmid insert of pSPE-2, lane MW, ladder of molecular weight markers Fig. 6 Eects of cellular polyamine status on spe-2 mRNA abundance.Total RNA was isolated from cultures of strain IC3 (aga) and the ODC-de®cient strain IC1894-53 (spe-1, aga), and subjected to Northern analysis using a 0.7-kb XhoI-BamHI fragment of pSP2 (spe-2, above), or a 1.2-kb SacI fragment of the N. crassa b-tubulin gene of pbT6 (tub, below) as probes.Lanes: 1, aga strain grown with 1 mM spermidine; 2, aga strain grown with 1 mM arginine (polyamine depleted); 3, spe-1, aga strain grown with 1 mM spermidine; 4, spe-1, aga grown with 1 mM arginine (severe polyamine depeletion) todes Ascaris suum and Onchocerca volvulus (Rathaur et al. 1988), and mammalian cells (Pegg and Williams-Ashman 1969) is stimulated by putrescine.We measured the eects of putrescine (up to 5 mM) on N. crassa AdoMetDC activity in desalted crude extracts (Fig. 7).The results show that putrescine is absolutely required for N. crassa AdoMetDC activity in vitro, with the K 0.5 being approximately 0.1 mM.

Processing of the N. crassa AdoMetDC proenzyme
In the N. crassa AdoMetDC coding region, both the amino acid sequences surrounding the glutamyl-serine bond ± the site of processing in the yeast and human AdoMetDC proenzymes ± and the amino acid residues required for stimulation by putrescine of processing of the human proenzyme, which lie elsewhere in the polypeptide, are conserved.This suggests that the N. crassa proenzyme is processed and that putrescine might stimulate processing.In this study we used a coupled in vitro transcription/translation system to con®rm that the N. crassa AdoMetDC enzyme is synthesized as a proenzyme and to determine the requirements, if any, for posttranslational processing of the proenzyme.We placed the spe-2 ORF downstream of the SP6 promoter in plasmid pSP6-SPE2 and synthesized the RNA and the encoded protein using the TNT SP6 assay system.We detected products corresponding to the proenzyme (predicted molecular weight 55 kDa) and the processed a subunit (44 kDa).We failed to detect the smaller (11 kDa) b subunit, perhaps due to its small size and the fact that it contains only two methionine residues.
Following a 30-min synthesis period, the majority of the N. crassa AdoMetDC appeared as the processed a subunit (Fig. 8A), and by 1 h after translation was inhibited with cycloheximide, almost all the proenzyme had disappeared.The N. crassa proenzyme, therefore, appears to be rapidly and autocatalytically processed to the mature form.The use of a shorter (10 min) synthesis period (Fig. 8B) revealed that approximately half the proenzyme was converted to the processed form 10 min after its synthesis.By 30 min after inhibition of translation, virtually all the proenzyme was processed, with a corresponding increase in the intensity of the band representing the mature a subunit.This is rapid compared to the human AdoMetDC: only 15% of this precursor is converted to the mature enzyme after 30 min in the absence of putrescine (Xiong et al. 1997).A previous study (Xiong et al. 1997) found that the TNT reticulocyte lysate, used in these experiments, contained insucient putrescine (<1 nM) to stimulate the processing of human AdoMetDC.Therefore, the rapid processing of the N. crassa proenzyme is unlikely to be caused by putrescine in the Inset: Lineweaver-Burk plot of the same data.The K 0.5 for putrescine, derived from the X-intercept, is approximately 100 lM Fig. 8A±C In vitro processing of the N. crassa AdoMetDC proenzyme.Using pSP6-SPE2 as a template, a coupled transcriptiontranslation reaction was allowed to proceed for 30 min (A) or 10 min (B, C) before terminating the reaction with 200 lg/ml cycloheximide.Samples were withdrawn periodically after termination for 60 or 120 min (top scale).The samples were subjected to SDS gel electrophoresis and visualized by autoradiography.Reactions were carried out in the absence (A, B) or presence (C) of 1 mM putrescine.The relative positions of molecular weight markers (in kDa) are indicated on the left.The small b subunit liberated in the processing reaction is not visible reaction mixture.Addition of 1 mM putrescine to the reaction failed to increase the rate of processing (Fig. 8C).The processing of the N. crassa proenzyme was also unaected by the addition of 1 mM MgCl 2 to the reactions (result not shown).These results suggest that the conservation of acidic amino acids, required for putrescine stimulation of processing in the human enzyme, is insucient to impart this requirement to the N. crassa proenzyme.

Discussion
Two noteworthy aspects of AdoMetDC of N. crassa have come to light in this work.First, a comparison of the structural gene sequence with other AdoMetDC genes and cDNAs reveals that it has a much longer amino acid sequence than any known to date.Initially, this suggested that introns might interrupt the N. crassa sequence.However, while a N. crassa cDNA was not retrieved and sequenced, the RT-PCR product and its restriction fragments did not dier in size from the corresponding genomic sequence.More interesting was the ®nding, occasioned by a successful search of the A. nidulans EST database, that the C-terminal region of AdoMetDC from this related ®lamentous fungus was quite similar in length to that of N. crassa AdoMetDC.The corresponding sequence of yeast (S. cerevisiae) diers from those of the two ®lamentous fungi.So far, the latter form a distinct and restricted class of AdoMetDCs.
Second, the processing of the N. crassa proenzyme appears to dier from that in mammals.Although cDNAs encoding the AdoMetDC proenzyme have been isolated from many organisms, the processing step and its stimulation by cellular cations has been characterized in only a few cases.The amino acid residues surrounding the site of processing are highly conserved in eukaryotes.The site of cleavage has been determined de®nitively for the proenzymes from human (Stanley et al. 1989), yeast (Kashiwagi et al. 1990), and potato (Xiong et al. 1997): processing occurs at a glutamyl-serine bond.It is therefore likely that processing of the N. crassa proenzyme occurs between the corresponding amino acid residues, Glu102 and Ser103.Although we have not demonstrated this directly, the size of the a subunit liberated in the in vitro processing reaction is consistent with this assumption.
Human AdoMetDC activity is activated by putrescine, but the activity of potato AdoMetDC, unlike that of N. crassa, is not (Xiong et al. 1997).Studies by Pegg and his collaborators have shown that putrescine both increases the rate of processing of the human Ado-MetDC proenzyme, and stimulates the catalytic activity of the mature enzyme (Pegg et al. 1998).They also found that mutation of amino acid residues distal to the processing site of the human proenzyme could slow or prevent cleavage of the proenzyme (Stanley and Pegg 1991;Stanley et al. 1994;Xiong et al. 1997).These mutations occurred within highly conserved regions, and at least four acidic residues (Glu11, Asp174, Glu178, and Glu256) were essential for stimulation of processing by putrescine (Stanley et al. 1994;Xiong et al. 1997).These acidic residues are conserved in the N. crassa proenzyme, as Glu44, Asp254, Glu258, and Glu398.The potato AdoMetDC proenzyme lacks the aspartic acid corresponding to the conserved residues in human (Asp-174) and N. crassa (Asp-254), and maximal rates of processing of the plant proenzyme do not require putrescine (Xiong et al. 1997).Based on a model in which the divalent putrescine bridges acidic residues in the proenzyme to bring about or stabilize a conformational change, Xiong et al. (1999) speculated that the absence of this aspartic acid might explain the lack of putrescinestimulated processing in the potato proenzyme, described in previous work (Xiong et al. 1997).Despite the conservation of all amino acid residues required for putrescine stimulation of processing in human Ado-MetDC in the N. crassa gene product, putrescine has no eect on the processing of the latter.It is noteworthy that insertions occur in the N. crassa sequence between the acidic amino acid residues required for putrescine stimulation of processing relative to the human sequence.Glu44 of the N. crassa proenzyme is separated from Asp254 and Glu258 by approximately 45 more residues than are the corresponding residues of the human proenzyme.Similarly, Asp254 and Glu258 are about 60 residues further away from Glu398 in the primary sequence than the corresponding human residues.In addition, the N. crassa proenzyme (and that of S. cerevisiae) has an extended N-terminal region when compared to the human AdoMetDC.These dierences may render ionic interactions between putrescine and the acidic residues unnecessary for optimal rates of processing.
The potato proenzyme and the N. crassa proenzyme are processed much more rapidly than the human proenzyme in vitro (Xiong et al. 1997).The mature a subunit appears almost immediately upon synthesis of the proenzyme in the case of potato, and the t 0.5 of the N. crassa proenzyme is about 10 min.Putrescine stimulation of these processing reactions would have little advantage.Possibly the processing of the human proenzyme and others like it may require a specially evolved stimulatory mechanism to compensate for their unusually slow rate of processing (t 0.5 80 min; Xiong et al. 1997).Only a systematic survey of putrescine stimulation of S-AdoMetDC proenzyme processing can answer this question.
The crystal structure of the mature human Ado-MetDC enzyme has been reported recently (Ekstrom et al. 1999).The ab monomer forms a novel a/b-sandwich fold comprising two eight-stranded antiparallel b sheets facing one another and ¯anked by a and 3 10 helices on the exterior of the b sheets.Comparison of the sequences of the human and N. crassa enzymes suggests that the additional amino acids in the N. crassa AdoMetDC might lie on the periphery of the monomer.
The insertion of the sequence Pro219±Glu250 of the N. crassa enzyme would occur in a sequence between b strands 8 and 9 of the human enzyme that connects the two facing b sheets of the monomer.The Lys282± Thr327 segment of N. crassa AdoMetDC would lie within the peripheral a-helix 8 of the human AdoMetDC monomer.Neither insertion, nor the large N-terminal extension, would necessarily impair the architecture of the residues surrounding the active site.
The catalytic activity of AdoMetDC from many eukaryotes is activated by putrescine.Three of the four conserved acidic amino acids involved in the putrescine stimulation of AdoMetDC proenzyme processing are also required for putrescine activation of the catalytic activity of the mature human enzyme (Stanley et al. 1994;Xiong et al. 1997).[The eects of mutation of the fourth residue (human Glu11) on putrescine activation could not be tested because it is required for catalytic activity; Stanley and Pegg 1991.]In the fungi S. cerevisiae and A. nidulans, putrescine stimulates or is required for AdoMetDC enzyme activity (Poso et al. 1975;Stevens et al. 1976).In the present work, we have demonstrated that in N. crassa, putrescine is an obligatory cofactor in the AdoMetDC reaction, and, in previous work, that the K m of the enzyme for S-adenosylmethionine decreases with increasing putrescine concentration (Pitkin and Davis 1990).Although putrescine appears not to be involved in processing of the proenzyme, the acidic residues conserved between the human and N. crassa proenzymes may nevertheless be required for the stimulation of catalytic activity.

Fig
Fig. 1A, B Partial genetic map of linkage group V and RFLP mapping of cosmid X2: B09.A Map of the spe-2±inl segment of N. crassa linkage group V, showing the relative positions of the spe-1 (ODC), spe-2 (AdoMetDC), am (NADP-glutamate dehydrogenase), his-1 (imidazole glycerolphosphate dehydratase) and inl (inositol synthase) genes.Map distances (in percent recombination) are shown below the line.B Distribution of an EcoRI polymorphism among progeny of a cross between Mauriceville (M) and Oak Ridge (O) parents (shown together with progeny, in parentheses), using probes speci®c for the genes involved.The distribution is shown for three of the markers shown in A and of the DNA insert of the X2: B09 cosmid, carrying the putative spe-2 gene.One of the progeny (dash) was not probed successfully in the case of the cosmid insert

Fig. 7
Fig. 7 Putrescine stimulation of the AdoMetDC reaction.The graph shows a plot of enzyme activity against putrescine concentration.Inset: Lineweaver-Burk plot of the same data.The K 0.5 for putrescine, derived from the X-intercept, is approximately 100 lM