Characterization of a Fungal Maleylacetoacetate Isomerase Gene and Identification of Its Human Homologue
1998; Elsevier BV; Volume: 273; Issue: 1 Linguagem: Inglês
10.1074/jbc.273.1.329
ISSN1083-351X
AutoresJosé M. Fernández‐Cañón, Miguel Á. Peñalva,
Tópico(s)Pancreatic function and diabetes
ResumoWe have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway.We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene.maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways.Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli.Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulansstrain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2,4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency. We have previously used Aspergillus nidulans as a fungal model for human phenylalanine catabolism. This model was crucial for our characterization of the human gene involved in alcaptonuria. We use here an identical approach to characterize at the cDNA level the human gene for maleylacetoacetate isomerase (MAAI, EC 5.2.1.2), the only as yet unidentified structural gene of the phenylalanine catabolic pathway. We report here the first characterization of a gene encoding a MAAI enzyme from any organism, the A. nidulans maiA gene.maiA disruption prevents growth on phenylalanine (Phe) and phenylacetate and results in the absence of MAAI activity in vitro and Phe toxicity. The MaiA protein shows strong amino acid sequence identity to glutathione S-transferases and has MAAI activity when expressed in Escherichia coli. maiA is clustered with fahA and hmgA, the genes encoding the two other enzymes of the common part of the Phe/phenylacetate pathways. Based on the high amino acid sequence conservation existing between other homologous A. nidulans and human enzymes of this pathway, we used the MaiA sequence in data base searches to identify human expressed sequence tags encoding its putative homologues. Four such cDNAs were sequenced and shown to be encoded by the same gene. They encode a protein with 45% sequence identity to MaiA, which showed MAAI activity when expressed in E. coli. Human MAAI deficiency would presumably cause tyrosinemia that would be characterized by the absence of succinylacetone, the diagnostic compound resulting from fumarylacetoacetate hydrolase deficiency in humans and fungi. Culture supernatants of an A. nidulansstrain disrupted for maiA are succinylacetone-negative but specifically contain cis and/or trans isomers of 2,4-dioxohept-2-enoic acid. We suggest that this compound(s) might be diagnostic for human MAAI deficiency. The catabolism of phenylalanine and tyrosine in humans is both of intrinsic and clinical interest. The enzymatic steps of this pathway were definitively established in the '50s by the work of Knox and colleagues (see Fig. 1 A; Ref. 1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar). However, two of its structural genes remained uncharacterized. We recently used a novel approach based on the development of a fungal model to characterize one of them (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar, 3Fernández-Cañón J.M. Peñalva M.A. J. Biol. Chem. 1995; 270: 21199-21205Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 4Fernández-Cañón J.M. Granadino B. Beltrán-Balero de Bernabé D. Renedo M. Fernández-Ruiz E. Peñalva M.A. de Córdoba S.R. Nat. Genet. 1996; 14: 19-24Crossref PubMed Scopus (217) Google Scholar). Here we report our successful application of this approach to the characterization of the other and address by reverse genetics the consequences of the corresponding enzyme deficiency in our model organism. An enzyme deficiency in any of the steps of this pathway causes in humans a known metabolic disease. For example, a deficiency in phenylalanine hydroxylase causes phenylketonuria (reviewed by Scriveret al. (5Scriver C.R. Eisensmith R.C. Woo S.L.C. Kaufman S. Annu. Rev. Genet. 1997; 28: 141-165Crossref Google Scholar)). Enzyme deficiencies in four other steps (those labeled as II, III, and VI in Fig. 1 A) cause different hypertyrosinemias (reviewed by Mitchell et al. (6Mitchell G.A. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A.L. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1994: 1077-1106Google Scholar)), and absence of homogentisate dioxygenase (IV, see Fig. 1 A) causes alkaptonuria (4Fernández-Cañón J.M. Granadino B. Beltrán-Balero de Bernabé D. Renedo M. Fernández-Ruiz E. Peñalva M.A. de Córdoba S.R. Nat. Genet. 1996; 14: 19-24Crossref PubMed Scopus (217) Google Scholar, 7La Du B.N. Zannoni V.G. Laster L. Seegmiller J.E. J. Biol. Chem. 1958; 230: 251-260Abstract Full Text PDF PubMed Google Scholar). Although the historical interest in the later is notable as it enabled Archibald Garrod to coin the term of "inborn error of metabolism" (8Garrod A.E. Lancet. 1902; 2: 1616-1620Abstract Scopus (465) Google Scholar, 9Garrod A.E. Lancet. 1908; 2: 73-79Google Scholar), the gene had not been characterized until recently (3Fernández-Cañón J.M. Peñalva M.A. J. Biol. Chem. 1995; 270: 21199-21205Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 4Fernández-Cañón J.M. Granadino B. Beltrán-Balero de Bernabé D. Renedo M. Fernández-Ruiz E. Peñalva M.A. de Córdoba S.R. Nat. Genet. 1996; 14: 19-24Crossref PubMed Scopus (217) Google Scholar, 10Granadino B. Beltrán-Valero de Bernabé D. Fernández-Cañón J.M. Peñalva M.A. Rodrı́guez de Córdoba S. Genomics. 1997; 43: 115-122Crossref PubMed Scopus (62) Google Scholar). Crucial for the isolation and characterization of this gene was our establishment of a fungal model for human phenylalanine catabolism based on the filamentous ascomyceteAspergillus nidulans (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar). We cloned its homogentisate dioxygenase gene (the first gene encoding this enzyme identified for any organism) and used its derived amino acid sequence as a probe to identify in similarity searches of the human expressed sequence, tag data base (EST) 1The abbreviations used are: EST, expressed sequence tag; HT1, human type 1 hereditary tyrosinemia; GSH, reduced glutathione; PhAc, phenylacetate; FAAH, fumarylacetoacetate hydrolase; HGO, homogentisate dioxygenase; MAAI, maleylacetoacetate isomerase; TMS, trimethylsilyl; kb, kilobase(s); GC, gas chromatography; MS, mass spectrometry; ORF, open reading frame; UTR, untranslated region. 1The abbreviations used are: EST, expressed sequence tag; HT1, human type 1 hereditary tyrosinemia; GSH, reduced glutathione; PhAc, phenylacetate; FAAH, fumarylacetoacetate hydrolase; HGO, homogentisate dioxygenase; MAAI, maleylacetoacetate isomerase; TMS, trimethylsilyl; kb, kilobase(s); GC, gas chromatography; MS, mass spectrometry; ORF, open reading frame; UTR, untranslated region. cDNAs encoding its human homologue (3Fernández-Cañón J.M. Peñalva M.A. J. Biol. Chem. 1995; 270: 21199-21205Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Type 1 hereditary tyrosinemia (HT1, hepatorenal tyrosinemia, McKusick 276700) is the most severe disease in human Phe catabolism, affecting liver, kidney, and peripheral nerves. HT1 patients surviving infancy develop chronic liver disease with a high incidence of hepatocellular carcinoma (6Mitchell G.A. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A.L. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1994: 1077-1106Google Scholar). HT1 results from fumarylacetoacetate hydrolase (FAAH) deficiency (11Lindblad B. Lindstedt S. Steen G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4641-4645Crossref PubMed Scopus (420) Google Scholar). It is generally accepted that fumarylacetoacetate and its spontaneous reaction product, succinylacetone (the diagnostic compound of the disease), are toxic due to their considerable reactivity with key cellular molecules (6Mitchell G.A. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A.L. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1994: 1077-1106Google Scholar, 11Lindblad B. Lindstedt S. Steen G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4641-4645Crossref PubMed Scopus (420) Google Scholar), and fumarylacetoacetate has been shown to be mutagenic in Chinese hamster cells (12Jorquera R. Tanguay R. Biochem. Biophys. Res. Commun. 1997; 232: 42-48Crossref PubMed Scopus (102) Google Scholar). In agreement with this, growth of an A. nidulans strain disrupted for the FAAH-encoding gene is prevented by phenylalanine even in the presence of an alternative carbon source (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar). succinylacetone is accumulated in culture supernatants of this strain, illustrating the equivalent consequences of a FAAH deficiency in humans and A. nidulans (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar). The clinical consequences of a MAAI (MAAI, EC 5.2.1.2; step V in Fig.1 A) deficiency in humans are largely unknown. It is predicted that this deficiency should also lead to HT1, as maleylacetoacetate has similar reactivity to fumarylacetoacetate (for example, see Ref. 13Overtuf K. Al-Dhalimy M. Tanguay R. Brantly M. Ou C. Finegold M. Grompe M. Nat. Genet. 1996; 12: 266-272Crossref PubMed Scopus (484) Google Scholar). By contrast, it is thought that it should not result in the presence of succinylacetone in plasma and urine, as the latter compound is likely to be formed from succinylacetoacetate resulting from in vivo reduction of maleyl and fumarylacetoacetate (6Mitchell G.A. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A.L. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1994: 1077-1106Google Scholar, 11Lindblad B. Lindstedt S. Steen G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4641-4645Crossref PubMed Scopus (420) Google Scholar). Succinylacetoacetate is efficiently degraded by FAAH (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar), and its hydrolysis would prevent succinylacetoacetone formation. Only one such succinylacetone-negative patient showing type 1 tyrosinemia with nondetectable levels of MAAI but normal levels of FAAH in liver has been described (14Berger R. Michals K. Galbraeth J. Matalon R. Pediatr. Res. 1988; 23 (abstr.): 328Google Scholar). Mammalian MAAI has been little studied since its original characterization (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar, 15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar, 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar), possibly due, among other possible reasons, to the instability of the substrate (6Mitchell G.A. Lambert M. Tanguay R.M. Scriver C.R. Beaudet A.L. Sly W. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1994: 1077-1106Google Scholar). The gene encoding MAAI has not been cloned from any organism, and it is therefore the only structural gene of the Phe/Tyr degradation pathway that remains uncharacterized, precluding the analysis of the molecular basis of succinylacetone-negative type I tyrosinemia. Here we successfully use our fungal model to identify cDNAs encoding human MAAI. The liver enzyme requires glutathione (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar, 15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar, 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar) as does the equivalent bacterial enzyme that has been purified to homogeneity (17Seltzer S. J. Biol. Chem. 1973; 248: 215-222Abstract Full Text PDF PubMed Google Scholar). Our characterization of fungal and human MAAI cDNAs revealed strong amino acid sequence identity of their derived protein sequences to glutathione S-transferases, in agreement with the proposed mechanism of the isomerization (18Morrison W.S. Wong G. Seltzer S. Biochemistry. 1976; 15: 4228-4233Crossref PubMed Scopus (9) Google Scholar). We also extend the work of Edwards and Knox (16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar) and demonstrate MAAI activity by an in vitrocomplementation assay using extracts from a recombinant fungal strain deficient for MAAI. Notably, we detected no succinylacetone in culture supernatants of this strain. A. nidulans strains carried markers in standard use (19Clutterbuck A.J. O'Brien S.J. 6th Ed. Genetic Maps. Locus Maps of Complex Genomes. 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 3.71-3.84Google Scholar). AbiA1 strain was used as a source of cDNA and wild type protein extracts. The biA1, methG1,ΔfahA strain has been described (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar). A biA1,methG1 strain was used as the wild type in growth tests. Standard media for A. nidulans (20Cove D.J. Biochim. Biophys. Acta. 1966; 113: 51-56Crossref PubMed Google Scholar) were used for strain maintenance, growth tests, and transformation. Culture conditions inducing high levels of expression of the fahA/maiA/hmgAgenes, which were routinely used to grow mycelia for protein extraction, have been described (21Fernández-Cañón J.M. Peñalva M.A. Anal. Biochem. 1997; 245: 218-221Crossref PubMed Scopus (27) Google Scholar). GenomicmaiA sequences were identified by Southern analysis of DNA from λEMBL4 clones carrying the fahA and hmgAgenes, using a subtracted cDNA probe representing genes induced by phenylacetate ("plus" probe) and a cDNA probe from glucose-grown mycelia ("minus" probe). A 2.5-kb EcoRI fragment contiguous to the fahA transcription unit (see Fig.1 B) showed strong differential hybridization to the plus probe. In addition, cDNA library screening using the F2 fragment (see Fig. 1 A) resulted in the isolation, in addition tofahA cDNA clones, of a second class of cDNA clones representing maiA transcripts. The cDNA library enriched in transcripts induced by phenylacetate and the subtracted plus cDNA probe have been described (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar, 3Fernández-Cañón J.M. Peñalva M.A. J. Biol. Chem. 1995; 270: 21199-21205Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The A. nidulansgenomic library was a standard λEMBL4 library constructed from the wild type strain. RNA isolation and Northern analysis followed (22Espeso E.A. Peñalva M.A. Mol. Microbiol. 1992; 6: 1457-1465Crossref PubMed Scopus (75) Google Scholar). Equal loading of the different samples was confirmed using an actin probe. Transformation followed Tilburnet al. (23Tilburn J. Scazzocchio C. Taylor G.G. Zabicky-Zissman J.H. Lockington R.A. Davies R.W. Gene. 1983; 26: 205-211Crossref PubMed Scopus (502) Google Scholar). For disruption of maiA we used a 4.2-kb linear DNA fragment in which the sequence betweenmaiA codons 140–226 had been replaced by a 3.2-kbXbaI fragment carrying argB +. A genomic fragment carrying maiA sequences from anXbaI site at position −133 (relative to the initiation codon) to an XhoI site at position +655 (relative to the stop codon) was subcloned in pBS-SK+ (Stratagene). Substitution of an internal 0.26-kb SalI-EcoRI fragment by the above 3.2-kb A. nidulans genomic fragment (whose XbaI ends had been previously converted toEcoRI and XhoI) removed maiA sequences between codons 140 and 226 to yield pBS-ΔMAI. The transforming fragment was isolated from this plasmid after digestion withXbaI and XhoI. Maleylacetoacetate, which is not commercially available, was synthesized enzymatically from homogentisate (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar, 15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar) using homogentisate dioxygenase from A. nidulans extracts or from Escherichia coli cells overexpressing the human enzyme. The procedure used to obtain mycelial protein extracts fromΔfahA, ΔmaiA, and wild type A. nidulans strains and the conditions for the homogentisate dioxygenase reaction have been described (21Fernández-Cañón J.M. Peñalva M.A. Anal. Biochem. 1997; 245: 218-221Crossref PubMed Scopus (27) Google Scholar). For in vitrocomplementation assays, the initial homogentisate concentration was 100–125 μm. Maleylacetoacetate formation was monitored spectrophotometrically at 330 nm. When the reaction reached a plateau (with usually more than 80% of the substrate converted to maleylacetoacetate), 150 μm reduced glutathione was added to allow the MAAI-dependent isomerization of maleylacetoacetate to fumarylacetoacetate (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar, 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar), which is then a substrate for FAAH. Complementation of ΔmaiA extracts was used to detect MAAI activity in crude lysates of E. colicells overexpressing fungal or human MAAI, as described in the corresponding figure legends. Maleylacetoacetate was synthesized in separate reactions using 250–500 μmsubstrate and an excess of recombinant human HGO. When the reactions reached a plateau, they were stopped and deproteinized after the addition of 0.1 vol of 10% metaphosphoric acid, incubation for 10 min in ice, and centrifugation at 13,000 × g for 10 min. The supernatants were neutralized with KOH to pH 7.5–8 and immediately used in in vitro reactions in the presence of wild type or mutant A. nidulans protein extracts. These reaction mixtures were essentially as for homogentisate dioxygenase but contained GSH as a cofactor for MAAI. Reactions were carried out for 15 min at room temperature, deproteinized with metaphosphoric acid, and neutralized with KOH as above. The absorption spectra of these samples were determined and compared with those of duplicate reaction mixtures for which the final neutralization step was omitted. High levels of protein expression were achieved using the pD1 vector (a gift of E. Espeso). This is a modified pET19b (Novagen) derivative that was engineered to introduce a single BamHI site allowing in-frame fusion of the desired coding region to an N-terminal His tag. Details of this vector will be described elsewhere. Proteins overexpressed in this system carry the sequence MGHHHHHHHHHHSSGHIDDDDKHMGS at their amino termini. The MaiA coding region was amplified using the following pair of primers (underlined sequences add or modify restriction sites): 5′-CGGGATCCCCCGCACCGGTCAAGATCTC-3′ (upper) and 5′-CGGAATTCAACACCTAAATTCCGTTGGTG-3′ (lower). The fusion protein contains the complete MaiA sequence with four further extra residues (PAPL) between the above N-terminal tag and the MaiA initiation methionine. The corresponding recombinant plasmid was denoted pD1::MaiA. The human MAAI coding region was amplified using EST 265310 (5′) as template and primers 5′-CAGGGATCCAAGCCCATCCTCTATTCC-3′ (upper) and 5′-CAGGAATTCGGAGCTAGGCCCTC-3′ (lower). The recombinant gene fused the above N-terminal tag to residues 5–216 of the protein. The corresponding plasmid was denoted pD1::HSMAAI. A pD1::HSHGO plasmid (a gift from M. C. Estébanez), driving high level expression of human HGO, will be described elsewhere. Recombinant plasmids were selected in E. coli DH1, purified, and transformed into E. coli BLB21(DE3)pLysS. Primary transformants were selected on LB plates containing ampicillin (100 μg/ml) and chloramphenicol (35 μg/ml) and directly used to inoculate LB liquid cultures that were grown at 37 °C untilA 600 reached 0.8–0.9 units. Expression of T7 RNA polymerase was induced after the addition of 0.4 mmisopropyl-1-thio-β-d-galactopyranoside and further incubation for 2.5 h. 0.5 ml samples were taken before and after induction, and bacteria were collected by centrifugation and resuspended in SDS-polyacrylamide gel electrophoresis loading buffer. Samples were boiled for 3 min before loading appropriate aliquots onto a 12% SDS-polyacrylamide gel alongside Bio-Rad wide-range protein markers. Proteins were detected by Coomassie staining. For preparation of protein extracts, bacteria from a 50-ml culture were collected by centrifugation, washed in 100 mm potassium phosphate buffer, pH 7.0, resuspended in 4 ml of the same buffer, and lysed by sonication. Lysates were clarified by centrifugation at 10,000 rpm and 4 °C for 20 min in an SS34 rotor. Protein concentrations were estimated by the Bradford assay (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). Fungal mycelia pregrown on 0.6% glucose (w/v) as the sole carbon source were transferred to appropriately supplemented minimal medium with 20 mmphenylacetate as the sole carbon source (see Ref. 21Fernández-Cañón J.M. Peñalva M.A. Anal. Biochem. 1997; 245: 218-221Crossref PubMed Scopus (27) Google Scholar) and incubated for 20 h at 37 °C. Culture filtrates were ether-extracted and derivatized with bis(trimethylsilyl)trifluoroacetamide as described (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar). TMS derivatives were analyzed by GC-MS in a fused silica capillary column SBP-1 (30 m × 0.25 mm; 0.2-mm film thickness) with a temperature program from 80 to 280 °C (4 °C/min), and a Q-MASS (Perkin-Elmer) mass detector. Identification of peaks was carried out by comparison of sample spectra with reference spectra from the NIST/EPA/NIH mass spectral data base. Genes and cDNAs were sequenced using a Dye Terminator Cycle sequencing kit (Perkin-Elmer) and TaqFS DNA polymerase with universal and custom primers. Sequencing reactions were resolved on an ABI Prism 377 automatic sequencer and analyzed with the ABI analysis software (Version 3.1). Genomic and cDNA versions of maiA and human EST cDNA clones 265310 (5′), 290219 (5′), 683733 (5′), and 52677(5′) encoding human MAAI were completely sequenced in both strands. cDNA clones of the IMAGE consortium (25Lennon G.G. Auffray C. Polymeropoulos M. Soares M.B. Genomics. 1996; 33: 151-152Crossref PubMed Scopus (1089) Google Scholar) were purchased from Genome Systems Inc., (St. Louis, MO). 2Details of EST libraries may be found inhttp://www-bio.llnl.gov/bbrp/image/humlib_info.html. The ascomycete fungusA. nidulans can use either Phe or PhAc as sole carbon source (Fig. 1 A). Both compounds were catabolized to homogentisate, which was then converted to fumarate and acetoacetate through the action of three enzyme activities, HGO, MAAI, and FAAH. We have previously reported that the A. nidulans fahA and hmgA genes encoding FAAH and HGO, respectively, are closely linked and divergently transcribed from a 414-base pair intergenic region. fahA and hmgAgene transcription is strongly inducible by PhAc (or its structural relatives) or Phe and partially repressible by glucose. Neither of these genes is expressed on glucose as the sole carbon source. No gene encoding MAAI has yet been characterized from any organism. Southern blot hybridization of λEMBL4 phage clones carrying thefahA and hmgA genes with a substracted cDNA probe representing transcripts induced by PhAc revealed the presence of a third linked gene strongly hybridizing to this probe. AsfahA and hmgA, this third gene, designatedmaiA, was not expressed on glucose. Clustering of genes encoding activities of the same catabolic pathway is not unusual inA. nidulans. Genomic and cDNA nucleotide sequencing of the region encoding this new transcript confirmed the presence of a third gene 3′ from fahA, transcribed in a tail-to-tail orientation (Fig. 1 B). The transcribed region contains an intron-less ORF encoding a putative 230-residue polypeptide (Fig.2) whose stop codon is 486 base pairs downstream from that of fahA. The nucleotide sequence of this three-gene cluster has been submitted to the DDBJ/EMBL/GenBankTM data bases under accession numberAJ001836. Data base searches revealed that the predicted product of maiAshows strong amino acid sequence identity to glutathioneS-transferases. For example, FASTA searches of the Swiss-Prot data base revealed that among the 30 protein sequences showing the highest alignment scores, 27 were glutathioneS-transferases (data not shown). Similar results were obtained after searching the conceptual translation of EMBL + GenBankTM nucleotide sequence data bases with TBLASTN. The highest FASTA score corresponded to glutathioneS-transferase 1 from Diantus caryophyllus, which showed 33.3% identity to MaiA in a 228-amino acid overlap including the complete sequence of both proteins (data not shown). The deduced molecular mass for MaiA (25,129 Da) is similar to the 25-kDa size of glutathione S-transferases. Northern analysis showed that, as determined for fahA and hmgA, transcription of maiA was induced by either Phe or PhAc and was absent on glucose or gluconeogenic carbon sources (Fig. 3), strongly suggesting thatmaiA was a gene for Phe/PhAc catabolism and that its clustering with fahA and hmgA reflected its involvement in the same catabolic pathway. The only as yet unidentified gene encoding an enzyme essential for both Phe and PhAc catabolism is that encoding MAAI. The likely mechanism of this enzyme involves transfer of enzyme-bound GSH to C2 of maleylacetoacetate (26Lee H.E. Seltzer S. Biochem. Int. 1989; 18: 91-97PubMed Google Scholar). Therefore, all the above data indicated that maiA might encode A. nidulans MAAI. To confirm this, we replaced by transformation the wild typemaiA gene by a mutant version in which the sequence encoding MaiA residues 140–226 had been substituted by a genomic DNA fragment containing the argB + gene (Fig.4 A). Transformants were selected in an argB2 background for arginine-independent growth and purified by repeated streaking of conidiospores on minimal medium lacking arginine. Two independent transformants showing the expected maiA replacement were selected after Southern analysis. Both showed an identical phenotype, being unable to grow on either phenylacetate or phenylalanine as the sole carbon source. This confirmed that maiA is a gene of the common part of the Phe/PhAc pathways. MAAI assays were carried out with protein extracts from mycelia of the disrupted strains grown on glucose and transferred to PhAc, which showed them to be deficient for MAAI activity (Fig.5). By contrast, these extracts showed normal levels of either FAAH or HGO (data not shown). Maleylacetoacetate can be synthesized in vitro by the homogentisate dioxygenase activity present in mycelial extracts from the wild type strain or from either a mutant ΔfahA strain (lacking FAAH (2Fernández-Cañón J.M. Peñalva M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9132-9136Crossref PubMed Scopus (56) Google Scholar)) or a mutant ΔmaiA strain (presumably lacking MAAI) and detected by its absorption at 330 nm (1Knox W.E. Methods Enzymol. 1955; 2: 287-300Crossref Scopus (31) Google Scholar, 15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar, 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar). In the absence of GSH, an obligate cofactor of MAAI, maleylacetoacetate is not isomerized to fumarylacetoacetate, thereby providing an enzymatic method to obtain the isomerase substrate (15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar, 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar). On addition of GSH, wild type extracts catalyze the isomerization of maleylacetoacetate to fumarylacetoacetate and the conversion of the latter to fumarate and acetoacetate. As neither of these two latter compounds shows the characteristic absorption of diketoacids in the near ultraviolet region, this coupled enzyme reaction can be monitored by the decrease of A 330 (see Fig. 5; Refs. 15Knox W.E. Edwards S.W. J. Biol. Chem. 1955; 216: 489-498Abstract Full Text PDF PubMed Google Scholar and 16Edwards S.W. Knox W.E. J. Biol. Chem. 1956; 220: 79-91Abstract Full Text PDF PubMed Google Scholar). Neither mutant extract alone would catalyze a decrease inA 330, as maleylacetoacetate (which would accumu
Referência(s)