Summary
The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bos CJ, Debets AJM, Slakhorst SM, Swart K (1989) Adenine and pyrimidine genes of Aspergillus niger and evidence for a seventh linkage group. Curr Genet 16:307–310
Clarke L, Carbon J (1978) Functional expression of cloned yeast DNA in E. coli: specific complementation of arginino-succinate lyase (argH) mutations. J Mol Biol 120:517–532
Cordewener JHG, Busink R, Boschloo J, Visser J, Bos CJ (1987) Development of biocatalysts with monooxygenase activity: Pathway and characterisation of enzymes of benzoate degradation in Aspergillus niger. In: Neijssel OM, Van der Meer RR, Luyben KChAM (eds) Proceedings 4th European Congress on Biotechnology, vol 3. Elsevier, Amsterdam, pp 366
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179:125–142
Gerritse K, Van Gorcom RFM, Fasbender M, Lange J, Schellekens MM, Zegers ND, Claassen E, Boersma W (1990) Specific immuno detection of benzoate-para-hydroxylase with antibodies raised against specific peptides. Biochem Biophys Research Commun 167:33–39
Goosen T, Bloemheuvel G, Gysler C, De Bie DA, Van den Broek HWJ, Swart K (1987) Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene. Curr Genet 11:499–503
Gotoh O, Tagashira Y, Iizuka T, Fujii-Kuriyama Y (1983) Structural characteristics of cytochrome P450: Possible location of the heme-binding cysteine in determined amino acid sequences. J Biochem 93:807–817
Hamid AB, Smith JE (1987) The involvement of cytochrome P-450 monooxygenase system in aflatoxin biosynthesis by Aspergillus flavus. J Ind Microbiol 2:137–142
Hinnebusch AG (1988) Novel mechanisms of translational control in Saccharomyces cerevisiae. Trends Genet 4:169–174
Hynes MJ, Corrick CM, King JA (1983) Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations. Mot Cell Biol 3:1430–1439
Kelly JM, Hynes MJ (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479
Klein P, Kanehisa M, DeLisi C (1985) The detection and classification of membrane-spanning proteins. Biochim Biophys Acta 815:468–476
MacRae WD, Buxton FP, Sibley S, Garven S, Gwynne DI, Davies RW, Arst Jr HN (1988) A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis. Gene 71:339–348
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Nebert DW, Gonzalez FJ (1987) P450 genes: Structure, evolution, and regulation. Annu Rev Biochem 56:945–993
Nebert DW, Nelson DR, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Kemper B, Levin W, Phillips IR, Sato R, Waterman MR (1989) The P450 superfamily: Updated listing of all genes and recommended nomenclature for the chromosomal loci. DNA 8:1–13
Nelson DR, Strobel HW (1988) On the membrane topology of vertebrate cytochrome P450 proteins. J Biol Chem 263:6038–6050
Nelson DR, Strobel HW (1989) Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam. Biochemistry 28:656–660
Pontecorvo G, Roper JA, Hemmons LJ, MacDonald KD, Buxton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Van den Hondel CAMJJ (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124
Punt PJ, Dingemanse MA, Jacobs-Meijsing BJM, Pouwels PH, Van den Hondel CAMJJ (1988) Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. Gene 69:49–57
Rao JKM, Argos P (1986) A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869:197–214
Reddy CC, Vaidyanathan CS (1975) Purification, properties and induction of a specific benzoate-para-hydroxylase from Aspergillus niger. Biochim Biophys Acta 384:46–57
Saloheimo M, Lehtovaara P, Penttilä M, Teeri TT, Ståhlberg J, Johansson G, Clacyssens M, Tomme P, Knowles JKC (1988) EGIII, a new endoglucanase from Trichoderma reesei: the characterisation of both gene and enzyme. Gene 63:11–21
Towbin H, Staehelin T, Goordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nail Acad Sci USA 76:4350–4354
Van Gorcom RFM, Punt PJ, Pouwels PH, Van den Hondel CAMJJ (1986) A system for the analysis of expression signals in Aspergillus. Gene 48:211–217
Van Gorcom RFM, Van den Hondel CAMJJ (1988) Expression analysis vectors for Aspergillus niger. Nucleic Acids Res 16:9052
Van Hartingsveldt W, Mattern IE, Van Zeijl CMJ, Pouwels PH, Van den Hondel CAMJJ (1987) Development of an homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Von Heijne G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690
Walsh C (1978) Chemical approaches to the study of enzymes catalyzing redox transformations. Annu Rev Biochem 47:881–931
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474
Yelton MM, Timberlake WE, Van den Hondel CAMJJ (1985) A cosmid for selecting genes by complementation in Aspergillus nidulans: selection of the developmentally regulated A locus. Proc Natl Acad Sci USA 82:834–838
Author information
Authors and Affiliations
Additional information
Communicated by W. Gajewski
Rights and permissions
About this article
Cite this article
van Gorcom, R.F.M., Boschloo, J.G., Kuijvenhoven, A. et al. Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: A member of a new gene family of the cytochrome P450 superfamily. Molec. Gen. Genet. 223, 192–197 (1990). https://doi.org/10.1007/BF00265053
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00265053