Harry C. M. Kester

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens

SummarySeveral nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed.The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline.They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides.

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory enzymes.

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, α-D-GalpA-([1→4]-α-D-GalpA)3-(1→4)-D-GalpA, is composed of five galacturonopyranose units (D-GalpA) linked by α-(1→4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein–oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC–galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the β elimination cleavage of polygalacturonic acid.

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

The Active Site Topology of Aspergillus niger Endopolygalacturonase II as Studied by Site-directed Mutagenesis*

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis inAspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K m and V max) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.

Molecular cloning, nucleotide sequence and expression of the gene encoding prepro-polygalacturonasell of Aspergillus niger

PolygalacturonaseII of Aspergillus niger was fragmented using CNBr and the NH2‐terminal fragment and another fragment were partially sequenced. The polygalacturonaseII (pgaII) gene was then isolated by using an oligonucleotide mixture based on the internal amino acid sequence as a probe. The nucleotide sequence of the pga II structural gene was determined. It was found that polygalacturonaseII is synthesized as a precursor having an NH2‐terminal prepro‐sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseII over‐producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctly processed and to be fully active.

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

SummaryThe nucleotide sequence of pelB, a member of the Aspergillus niger pectin lyase multigene family, has been determined. The pelB gene product, PLB, shares 65% amino acid identity with pectin lyase A (PLA) and 60% with pectin lyase D (PLD). Although growth of pelB multicopy transformants on pectin-containing media results in elevated pelB mRNA levels, pectin lyase B (PLB) is barely detectable. This is probably due to degradation of PLB by acid proteases, since multicopy transformants grown on pectin medium with a high concentration of phosphate, leading to a less rapid decline in pH, secrete detectable amounts of PLB. To produce PLB in high amounts under conditions where few other extracellular enzymes are present, we tried two strategies. Firstly, heterologous expression of the pelB gene in A. nidulans, and secondly, expression of the pelB gene under control of the constitutive A. niger pki promoter.

Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties

We isolated and characterized a new type of endopolygalacturonase (PG)‐encoding gene, pgaD, from Aspergillus niger. The primary structure of PGD differs from that of other A. niger PGs by a 136 amino acid residues long N‐terminal extension. Biochemical analysis demonstrated extreme processive behavior of the enzyme on oligomers longer than five galacturonate units. Furthermore, PGD is the only A. niger PG capable of hydrolyzing di‐galacturonate. It is tentatively concluded that the enzyme is composed of four subsites. The physiological role of PGD is discussed.

Subsite Mapping of Aspergillus niger Endopolygalacturonase II by Site-directed Mutagenesis

To assess the subsites involved in substrate binding in Aspergillus niger endopolygalacturonase II, residues located in the potential substrate binding cleft stretching along the enzyme from the N to the C terminus were subjected to site-directed mutagenesis. Mutant enzymes were characterized with respect to their kinetic parameters using polygalacturonate as a substrate and with respect to their mode of action using oligogalacturonates of defined length (n = 3–6). In addition, the effect of the mutations on the hydrolysis of pectins with various degrees of esterification was studied. Based on the results obtained with enzymes N186E and D282K it was established that the substrate binds with the nonreducing end toward the N terminus of the enzyme. Asn186 is located at subsite −4, and Asp282 is located at subsite +2. The mutations D183N and M150Q, both located at subsite −2, affected catalysis, probably mediated via the sugar residue bound at subsite −1. Tyr291, located at subsite +1 and strictly conserved among endopolygalacturonases appeared indispensable for effective catalysis. The mutations E252A and Q288E, both located at subsite +2, showed only slight effects on catalysis and mode of action. Tyr326 is probably located at the imaginary subsite +3. The mutation Y326L affected the stability of the enzyme. For mutant E252A, an increased affinity for partially methylesterified substrates was recorded. Enzyme N186E displayed the opposite behavior; the specificity for completely demethylesterified regions of substrate, already high for the native enzyme, was increased. The origin of the effects of the mutations is discussed.

Inversion of configuration during hydrolysis of α-1,4-galacturonidic linkage by three Aspergillus polygalacturonases

Endopolygalacturonases I and II (PGI and PGII) of Aspergillus niger and an exopolygalacturonase (ExoPG) of A. tubingensis were investigated to reveal the stereochemistry of their hydrolytic action. Reduced pentagalacturonic acid (pentaGalU‐ol) and reduced trigalacturonic acid (triGalU‐ol) were used as non‐reducing substrates for the enzymes. The configuration of the reducing ends in the products formed in D2O reaction mixtures was followed by 1H‐NMR spectroscopy. It has been unambiguously established that primary cleavage of pentaGalU‐ol by both PGI and PGII leads to diGalU‐ol and the β‐anomer of triGalUA. The primary products of hydrolysis of triGalU‐ol by ExoPG were diGalU‐ol and the β‐anomer of GalUA. Thus, all three Aspergillus polygalacturonases belong to the so‐called inverting glycanases, i.e. they utilize the single displacement mechanism of hydrolysis of the glycosidic linkage.