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

Abstract

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3±7).Apparent V max values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mkat´mg ±1 , 36.5 mkat´mg ±1 and 415 nkat´mg ±1 for endopolygalacturonases I, II and C, respectively. K m values were , 0.15 mg´mL ±1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes.Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated D4,5-unsaturated oligogalacturonates of degree of polymerization 4±8. Whereas endopolygalacturonase I showed a strong preference for generating the D4,5-unsaturated dimer, with endopolygalacturonase II the D4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the D4,5-unsaturated dimer and trimer were observed, although in different ratios.Keywords: Aspergillus niger; endopolygalacturonase; kinetics; processivity; subsites.Saprophytic and plant pathogenic fungi and bacteria produce a vast array of enzymes capable of degrading the complex carbohydrate structures present in the plant cell wall. The most complex carbohydrate, and one of the major constituents of the middle lamella of the plant cell wall, is pectin. Owing to its complex structure, many enzymes are involved in the complete breakdown of pectin. Among these enzymes are pectin methylesterases, pectin and rhamnogalacturonan acetylesterases, pectate, pectin and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases and polygalacturonases. The polygalacturonases [poly(1,4-a-d-galacturonide) glycanohydrolase, EC 3.2.1.15] hydrolyze the a-1,4 glycosidic bonds between adjacent a-dgalacturonic acid residues and are thought to act specifically on the homogalacturonan or`smooth' part of the pectin molecule.At present numerous genes encoding both exo-and endo-acting polygalactu...

Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates

“…DP PME activity (units:mg the hexagalacturonate was 100-fold higher than that with the trigalacturonate (10.7 and 0.1 units:mg −" respectively). This indicates that the substrate-binding site of PME is composed of multiple subsites, which is a common feature of pectindepolymerizing enzymes [12,14]. Oligomers with a higher DP interact with more subsites during substrate binding.…”

Substrate Recognition and Hydrolysis by a Family 50 exo-β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans

“…Davies and Henrissat (25) forwarded the observation that enzymes possessing a tunnel-shaped catalytic groove typically act as processive enzymes. Processivity has been described for several types of glycoside hydrolases, including amylases, ␤-glucanases, cellulases, chitinases, carrragenases, and polygalacturonases (57)(58)(59)(60)(61)(62). The particular topology of such a catalytic site has been shown to be a key factor in the efficiency of microcrystalline polysaccharide degradation (63)(64)(65).…”

Subsite Mapping of Aspergillus nigerEndopolygalacturonase II by Site-directed Mutagenesis

“…Three conserved aspartate residues (Asp 180 , Asp 201 , and Asp 202 in PGII) appeared critical for catalysis. Asp 180 , with the assistance of Asp 202 , was proposed to act as a base to activate the bound water molecule, and Asp 201 was tentatively identified as the general acid that protonates the leaving group (12).Each of the A. niger PGs has different specific kinetic parameters on polygalacturonic acid and a specific mode of action (4,7,8). Furthermore, the enzymes display a different tolerance or preference for partially methylesterified substrates.…”

“…Each of the A. niger PGs has different specific kinetic parameters on polygalacturonic acid and a specific mode of action (4,7,8). Furthermore, the enzymes display a different tolerance or preference for partially methylesterified substrates.…”

“…For many microbial enzymes involved in pectin degradation, corresponding genes have been cloned and sequenced. In Aspergillus niger, a family of seven highly homologous genes encoding endopolygalacturonases (PGs) 1 (EC 3.2.1.15), has been discovered (1-4), and six of them have been overexpressed (4,7,8). The corresponding enzymes, called PGI, PGII, PGA-C, and PGE, which belong to family 28 of the glycosyl hydrolases (5,6) and catalyze the random hydrolysis of 1,4-␣-D-galactosiduronic linkages in the smooth regions of pectin, have been biochemically studied (4,7,8).…”