pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger

Abstract

In the present study, the molecular and basic biochemical characterization of endopolygalacturonase E, the fourth Aspergillus niger N400 endopolygalacturonase, is reported. The entire endopolygalacturonase E gene consists of 1293 bp interrupted by three short introns (50, 50, and 59 bp, respectively) as concluded from the cDNA sequence. The deduced amino acid sequence comprises 378 residues that include 39 Nterminal amino acids of the prepropeptide. The calculated M r and pI of the mature protein are 35 584 and 3.6, respectively.Compared with other endopolygalacturonases from A. niger N400, the mature protein endopolygalacturonase E has the highest sequence identity with endopolygalacturonase C (77.6 %) followed by endopolygalacturonase I (57.6 %) and endopolygalacturonase II (54.3 %).For overproduction of endopolygalacturonase E, an A. niger multicopy strain was used that was transformed with a promoter gene fusion construct that directs expression from the glycolytic A. niger pyruvate kinase promoter. The enzyme was purified and characterized as an endopolygalacturonase based on product analysis after polygalacturonate hydrolysis and on bond cleavage frequencies of oligogalacturonates of different degree of polymerisation (n ϭ 2Ϫ7). The pH optimum was 3.8. The K m and V max for polygalacturonate hydrolysis were 2.5Ϯ 0.4 mg · ml Ϫ1 and 1.3Ϯ 0.2 µkat · mg Ϫ1 , respectively. A subsite map was calculated by the combination of the methods of Suganuma et al.

Characterization of a novel endopolygalacturonase fromAspergillus nigerwith unique kinetic properties

“…The same rate increase was also recorded for r(GalpA) 5 and r(GalpA) 6 which also cover subsite 33. Moreover, binding of r(GalpA) 6 at subsite 34 appears not favorable, this in contrast to PGI, II, A, C and E [4,6]. From these data and the product progression on polygalacturonic acid, it is tentatively concluded that PGD is composed of only four functional subsites stretching from 33 to +1.…”

“…This learned that 66% of the binding modes of (GalpA) 5 in (GalpA) 4 3GalpA mode resulted in processive attack. The decrease of hydrolysis observed for (GalpA) 5À7 compared to (GalpA) 4 may be caused by the temporal tying up of the enzyme in the unproductive enzyme-product complex prior to the multiple attack.…”

“…(GalpA) 2 formation from (GalpA) 5 served as the identi¢er of the ¢rst event in that particular mode and thus for the calculation of that particular frequency (58%). From this, the frequency of the binding mode that in a ¢rst event would result in the formation of GalpA and (GalpA) 4 logically followed (42%). Next, the difference between the amount of (GalpA) 2 and (GalpA) 3 served for the calculation of the degree of processivity.…”

“…At both substrate concentrations, the strongest rate increase (15-fold) was recorded upon occupying subsite 33, viz. (GalpA) 4 . The same rate increase was also recorded for r(GalpA) 5 and r(GalpA) 6 which also cover subsite 33.…”

“…A ratio plot according to 5 . Due to the fact that processive behavior was observed already on (GalpA) 5 and the strong preference for hydrolysis of (GalpA) 4 at the ¢rst glycosidic linkage from the reducing end allowed for the calculation of the initial binding modes for (GalpA) 5 as presented in Table 1. (GalpA) 2 formation from (GalpA) 5 served as the identi¢er of the ¢rst event in that particular mode and thus for the calculation of that particular frequency (58%).…”