Section Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands1
Author for correspondence: Peter Schaap. Tel: +31 317 485142. Fax: +31 317 484011. e-mail: peter.schaap{at}algemeen.mgim.wag-ur.nl
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ABSTRACT |
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Keywords: metallopeptidase
Abbreviations: pNA, p-nitroanilide
The EMBL accession number for the sequence reported in this paper is AJ292570.
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INTRODUCTION |
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The majority of aminopeptidases belong to the M1 family of peptidases; they are metalloenzymes (Van Wart, 1996 ) which require zinc for enzymic activity and share the zinc binding motif HEXXH (Jongeneel et al., 1989
; Hooper, 1994
).
The evolutionary tree of the M1 family of metallo-aminopeptidases (Barret et al., 1998 ) shows that this family can be divided into three main groups. Two groups, the aminopeptidase N group and the leukotriene A4 hydrolase group, have been fully characterized. The remaining aminopeptidases within the M1 family are grouped together, mainly because they share a high sequence similarity. However, peptidases from this group that have been biochemically characterized differ considerably in their characteristics.
Two yeast enzymes from the M1 group have been actively studied (Hirsch et al., 1988 ; Garcia-Alvarez et al., 1991
; Caprioglio et al., 1993
). In the case of industrially used filamentous fungi, like Aspergillus spp., only aminopeptidase activities from Aspergillus oryzae have been described. A. oryzae produces at least seven aminopeptidase activities (Nakadai & Nasuno, 1977
) of which four have been purified (Nakadai & Nasuno, 1977
; Nakadai et al., 1973a
, b
, c
) and one has been cloned (Blinkovsky et al., 2000
).
Our aim is to characterize the pathways involved in protein catabolism in Aspergillus niger. So far seven endopeptidases (see van den Hombergh et al., 1997 , and references therein), one maturase (Jalving et al., 2000
) and two carboxypeptidases (van den Hombergh et al., 1994
; Dal Degan et al., 1992
; Svendsen & Dal Degan, 1998
) of A. niger have been cloned and characterized. To date no aminopeptidases of A. niger have been cloned or characterized. Here we present the characterization of the apsA gene encoding an intracellular zinc aminopeptidase of A. niger and the characterization of the purified enzyme.
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METHODS |
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Cloning of apsA.
An apsA PCR product was generated using degenerate primers based on regions conserved between the metallo-aminopeptidase amino acid sequence of Saccharomyces cerevisiae (accession nos P37898, P32454), Mus musculus (AAC52409), Homo sapiens (P15144), Oryctolagus cuniculus (S07099), Haemonchus contortus (CAA63897) and Rattus norvegicus (AAB38021).
The conserved peptide sequences GAMENWG and HELAHQW were used to design a forward primer, 5'-GGIGCNATGGARAAYTGGGG-3', and a reverse primer, 5'-AAICCRAACCAYTGRTGNGC-3', respectively (standard IUB-IUPAC symbols are used to indicate the nucleotide mixtures, I denotes inosine). A standard PCR was performed on genomic DNA of A. niger N402 using an equal amount of both primer mixtures and an annealing temperature of 50 °C. The amplified product was cloned in pGEM-T (Promega) and sequence analysis followed. The PCR product was used as a probe in the screening of a EMBL4 genomic library of A. niger N400 by standard methods to obtain the apsA gene. Three phages were isolated and from one positive phage a 1·7 kb EcoRIBamHI fragment and a partially overlapping 2·7 kb SalI fragment were subcloned in pUC19 and sequenced over both strands. cDNA of apsA was generated by RT-PCR, using the enhanced avian RT-PCR kit of Sigma, according to the suppliers instructions.
Protein and nucleotide sequence analyses were done with the program DNASTAR (Lasergene). The BLAST algorithm (Altschul et al., 1997 ) was used to search the public databases. Multiple alignments were made with CLUSTAL X (Jeanmougin et al., 1998
).
Plasmid construction and overexpression of ApsA.
The 1·7 kb EcoRIBamHI fragment and the partially overlapping 2·7 kb SalI fragment were merged, resulting in pIM4102 (Fig. 1). Plasmids pIM4102 and pGW635, which contain the A. niger pyrA gene, were used to co-transform A. niger NW219 according to Kusters-van Someren et al. (1991)
. PyrA+ transformants were screened for enhanced aminopeptidase activity in cell extracts. For this, ground mycelium was extracted with 100 mM sodium/potassium phosphate buffer at pH 7·2 and clarified by centrifugation (10000 g for 15 min at 4 °C). Aminopeptidase activity in these cell extracts was determined as described below and protein concentrations were determined by the bicinchoninic acid method as described by the supplier (Sigma).
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Biochemical characterization of ApsA.
Aminopeptidase activity was determined as described by Atlan et al. (1994) . A range of amino acids coupled to p-nitroanilide (pNA) were used as substrate. Standard conditions were 2 mM substrate in 67 mM sodium/potassium phosphate buffer at pH 7·2 and 30 °C. One unit of enzyme activity is defined as the amount of enzyme that produces 1 µM pNA min-1 L-pNA, P-pNA, R-pNA, F-pNA, A-pNA, M-pNA and K-pNA were obtained from Sigma and V-pNA, G-pNA, I-pNA and E-pNA were obtained from Bachem. The optimal pH for enzymic activity was determined using McIlvaine buffers at pH values ranging from 3 to 8, 200 mM HEPES plus 300 mM NaCl at pH values from 7·2 to 8·5 and 50 mM Tris/glycine buffer at pH values from 8 to 10. The pH stability of ApsA was tested by preincubation of the purified enzyme in McIlvaine buffer of different pH values ranging from 2·2 to 8 at 30 °C for 90 min followed by the standard enzyme reaction. The temperature stability of ApsA was tested by preincubation of the purified enzyme at 0, 30, 40, 50 and 60 °C for 60 min in sodium/potassium phosphate buffer, pH 7·2, followed by the standard enzyme reaction. Here the 0 °C preincubated sample was used as a reference to calculate the residual activity.
The effect of the protease inhibitors bestatin, 1,10-phenanthrolin, EDTA, EGTA, PMSF, tosyl phenylalanyl chloromethyl ketone (TPCK), tosyl lysyl chloromethyl ketone (TLCK), leupepstatin and iodoacetamide on enzymic activity was measured in sodium/potassium phosphate buffer at pH 7·2. The purified enzyme was preincubated with the respective compound for 30 min at 30 °C. After the preincubation period substrate was added, followed by the standard enzyme assay. The metal ion requirement of ApsA for activity was tested by preincubating the enzyme with EDTA, EGTA or 1,10-phenanthrolin for 15 min at 37 °C or for 17 h at 4 °C.
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RESULTS AND DISCUSSION |
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The encoded protein is similar to the M1 family of metallo-aminopeptidases (Barret et al., 1998 ). ApsA has highest similarity with LAPI (encoded by APE2), (Garcia-Alvarez et al., 1991
) and AAPI (encoded by AAPI), (Caprioglio et al., 1993
), both from S. cerevisiae (53·3 and 50·9% overall identity, respectively; Fig. 1
). The region of highest identity is found at the N-terminal part of the enzyme. The encoded protein contains the signature sequence of the M1 family of zinc peptidases (HEXXH) (Jongeneel et al., 1989
; Hooper, 1994
) and this sequence was found in a region that is most conserved between ApsA, AAPI and LAPI (Fig. 1
).
Northern analysis of A. niger N402 grown on several carbon and nitrogen sources showed that the apsA messenger levels were independent of the carbon or nitrogen source used (results not shown). The yeast aminopeptidase genes APE2 and AAPI are also constitutively expressed, although yeast AAPI mRNA, which is present during all phases of growth, is reported to be more abundantly expressed in exponentially growing yeast cells (Caprioglio et al., 1993 ).
Since ApsA does not contain a known secretion signal nor a known organellar targeting signal and since apsA transcript levels are apparently not influenced by the carbon or nitrogen sources tested, we conclude that ApsA is, like LAPI and AAPI, located in the cytosol.
Overexpression of the gene encoding ApsA in A. niger
A. niger strain NW219 was transformed with plasmid pIM4102. Nine transformed strains were further analysed for the occurrence of multiple integrations of the plasmid in the genome. Southern analysis showed that transformants 7 (Tr7) and 8 (Tr8) have the highest copy numbers of the integrated plasmid (Fig. 2). Messenger levels of apsA of Tr7 and Tr8 were compared to the messenger level of the wild-type strain by Northern analysis. Scanning of Northern blots revealed that compared to the wild-type strain both transformants have at least a tenfold increased messenger level of the correct size compared to the wild-type (Fig. 2
).
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Purification and biochemical properties of the enzyme
ApsA was purified from a cell extract of Tr7, resulting in an enzyme preparation with a specific activity of 12 U mg-1. The final yield was 5% (Table 1). The low yield after the first step is probably due to ammonium sulphate precipitation.
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Fraction 11 contained the highest activity and was used for further characterization of the enzyme (Fig. 3). The enzyme efficiently hydrolyses K-pNA and R-pNA, suggesting that the enzyme prefers basic amino acids at the N-terminal end of the substrate. The Km and Kcat for K-pNA and L-pNA were 0·17 mM and 0·49 µkat mg-1, and 0·16 mM and 0·31 µkat mg-1, respectively. The enzyme also hydrolyses M-pNA and has some activity towards A-pNA and F-pNA (Table 2
).
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In the cell extracts of Tr7 and Tr8, the highest increase in activity was found towards K-pNA and R-pNA, followed by L-pNA. However, no increase in activity towards F-pNA was found. The purified ApsA, however, shows some activity towards F-pNA. This suggests that other, more specific F-pNA hydrolysing activities present in the cell extract are dominant over a relatively small increase in the two transformants.
The enzyme is active between pH 5 and 9 (Fig. 4). The optimal pH is between 7·5 and 8·0 which is close to the pH of the Aspergillus cytosol of pH 7·8 (Hesse et al., 2000
). This slightly differs from the optimal pH of LAPI (pH 7·5) (Trumbly & Bradley, 1983
). The optimal pH of AAPI has not been reported.
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Several possible inhibitors were tested (Table 3). The three serine protease inhibitors tested had no effect on the enzyme activity. Iodoacetamide also did not have any effect on the activity of ApsA, indicating that the enzyme is not a cysteine aminopeptidase. The aminopeptidase inhibitor bestatin was able to inhibit ApsA activity. 1,10-Phenanthrolin was also able to inhibit the activity of ApsA, probably by chelating the metal ion bound in the enzyme. Surprisingly, the metal chelators EDTA and EGTA were not able to reduce the activity after a pre-incubation of 30 min at 30 °C. A 17 h incubation at 4 °C with EDTA or EGTA was necessary to reduce the activity of ApsA completely. This suggests that the metal ion is bound strongly to the ApsA enzyme.
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In this study we cloned and characterized the first aminopeptidase gene of the M1 family of aminopeptidases from a filamentous fungus. We also determined the general biochemical characteristics of the encoded enzyme. The substrate specificity is different from that of the yeast enzymes; lysine and not leucine, arginine or alanine is preferred at the N-terminal position. This genetic and biochemical characterization will enable further studies for the understanding of the in vivo roles of the aminopeptidases in degradation of (imported) peptides.
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ACKNOWLEDGEMENTS |
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Received 12 February 2001;
revised 9 April 2001;
accepted 1 May 2001.