The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes

Abstract

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases T and I1 (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergilh nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A . niduluns transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pguC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intronlexon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A . niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to rcpresent a binding site for a regulatory protein as it shows a high similarity to the yeast CYCl upstream activation site recognized by the HAP2/3/4 activation complex.Polygalacturonases are involved in the degradation orpectin, a complex polysaccharide that is primarily found in the middle lamella and primary cell wall of higher plants [I]. Polygalacturonases as well as other pectolytic enzymes are produced by many organisms (reviewed in [2]), and microbial pectolytic enzymes have been extensively studied in relation to plant pathogenesis. The molecular biology of the bacterial enzymes, especially the pectate lyases of Erwiniu, is well developed compared to that of the pectolytic enzymes of fungal pathogens [3, 41. Recently, genomic or cDNA clones coding for pectinesterase [5], pectin lyases [6, 71, pectate lyase [8] and polygalacturonases [9 -121 have been obtained from Aspei-gillus species, which are considered to be saprophytes. The availability of commercial pectinase preparations derived from Aspergillus niger has contributed to the rapid progress in gene Correspondence to J. Visser, Section

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C

“…Transformants were analysed for endopolygalacturonase production by SDS/PAGE (results not shown). No bands were visible in the lane that contained a control sample from the host strain itself, A. niger NW156, but for each fusion several transformants showed a band of the expected size [1] namely < 58 kDa for endopolygalacturonase I, 38 kDa for endopolygalacturonase II and 61 kDa for endopolygalacturonase C (data not shown). For endopolygalacturonase II the observed molecular mass agrees well with the 34 963 Da calculated from the deduced amino acid sequence.…”

“…Both (GalpA) 4 and (GalpA) 5 originate from binding of the substrate in the mode that generates these oligomers in a first hydrolysis event but also arise from the processive attack starting at the reducing end. From the bond-cleavage frequencies for endopolygalacturonase C (Table 3), it is clear that the high amount of (GalpA) 1 in Fig. 3C not only originated from processivity but also from the very strong preference for hydrolysing oligomers at the first glycosidic linkage.…”

“…For endopolygalacturonase C acting on (GalpA) 7 , similar results were found (not shown). For endopolygalacturonase I, using (GalpA) 7 as substrate, high amounts of (GalpA) 1 and hardly any (GalpA) 6 were observed. Similarly, using (GalpA) 8 as substrate, hardly any (GalpA) 7 and (GalpA) 6 were detected whereas large amounts of (GalpA) 1 and (GalpA) 2 accumulated (not shown).…”

“…Aspergillus niger was shown to harbor a complete family of endopolygalacturonase-encoding genes, [1] all of which have now been cloned, sequenced and individually overexpressed [2±5] (L. Par Ïenicova  and J. Visser, unpublished data). The presence of such a family of genes raises intriguing questions.…”

Characterization of a novel endopolygalacturonase fromAspergillus nigerwith unique kinetic properties

“…In the saprophytic fungus Aspergillus niger, a multigene family encoding endopolygalacturonases (PGs) was identi¢ed [1]. Thus far, six out of seven members of the PG-encoding gene family have been sequenced [1^5] and the corresponding enzymes biochemically characterized [4^6].…”