Oskar Markovič

Crystal Structure of Plant Pectin Methylesterase

Pectin is a principal component in the primary cell wall of plants. During cell development, pectin is modified by pectin methylesterases to give different properties to the cell wall. This report describes the first crystal structure of a plant pectin methylesterase. The β‐helical structure embodies a central cleft, lined by several aromatic residues, that has been deduced to be suitable for pectin binding. The active site is found at the center of this cleft where Asp157 is suggested to act as the nucleophile, Asp136 as an acid/base and Gln113/Gln135 to form an anion hole to stabilize the transition state.

An essential tyrosine residue of Aspergillus polygalacturonase

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine‐reactive agents tested, acetic anhydride, N‐acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3–9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

PectinaseAspergillus sp. polygalacturonase: Multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases

Nine forms ofAspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with freea-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from otherAspergillus species (72–75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66–71% residue differences toward theErwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties

The glycoprotein character of multiple forms of Aspergillus polygalacturonase.

Comparisons of known primary structures of polygalacturonases show that extent and localization of potential N-glycosylation sites differ. Some sites are similar in position and adjacent to strictly conserved residues at the potential active site. The presence of N-acetylglucosamine and mannose in the molecules of two homogeneous, major Aspergillus sp. polygalacturonase forms was confirmed by IR spectroscopy. The purification method, based on interaction of the carbohydrate part with concanavalin A immobilized on chlorotriazine bead cellulose, was optimized. Deglycosylation with N-glycosidase F under denaturating and nondenaturating conditions led to molecular mass decreases followed by complete inactivation of the polygalacturonase enzyme activity. These results show the importance of glycosylation in these protein forms, while the comparative patterns establish both variability and some similarities in overall glycosylation architectures.

The action of tomato and Aspergillus foetidus pectinesterases on oligomeric substrates esterified with diazomethane

Abstract The action of tomato and A. foetidus pectinesterases on oligo( d -galactosiduronic acids) partially esterified with diazomethane was studied. The pentamer was found to be the shortest substrate at which de-esterification catalyzed by tomato pectinesterase occurred, whereas A. foetidus pectinesterase was able to cause attack on the dimer. With increasing degree of polymerization of the esterified oligomers, the reaction rates increased for both enzymes. Differences were, however, found in the relative activities towards the oligomeric substrates, as well as in the extent of their de-esterification. From these results, it is apparent that the two enzymes studied differ in both the size and the character of the active site.

Chromogenic substrate for endo-polygalacturonase detection in gels

Abstract A simple, sensitive zymogram technique for the detection of endo-polygalacturonase (EC 3.2.1.15) in gel slabs after electrophoresis or isoelectrofocusing was developed. This technique employs a new chromogenic substrate prepared by coupling D -galacturonan DP 10 with Ostazin Brilliant Red S-5B dye. The detection of multiple forms of endo-polygalacturonase is based on the selective removal of depolymerized dyed substrate from the agar replicas by a solvent system that does not solubilize non-degraded dyed D -galacturonan DP 10 present in agar gel replicas.

Use of cross-linked hydrolysed starch in gel electrophoresis

Abstract A gel prepared from cross-linked hydrolysed starch preserves the optimal electrophoretical separation properties and exhibits better mechanical features than those of hydrolysed starch. Its improved mechanical strength and elasticity make it more suitable for slicing and for multiple simultaneous detections of the same sample.

MODIFICATION OF TOMATO AND ASPERGILLUS NIGER PECTINESTERASES WITH DIETHYL PYROCARBONATE

The role of histidine residues in pectinesterases was evaluated by monitoring the sensitivity to modification with diethyl pyrocarbonate in the tomato andAspergillus niger enzymes. Different and incomplete losses of enzyme activity were obtained. Inactivation of the enzymes was proportional to the histidine content (two in the tomato T1 form, six in theAspergillus form), suggesting that accessible histidine residues do not have active-site functions in these pectinesterases, but contribute to the overall structural stability. Lack of His roles in common between the enzyme forms is in agreement with the structures of pectinesterases having no conserved His residues.