Publications

• Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris.

  Mertens J.A., Bowman M.J.

  Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae ... >> More

  Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40 °C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 μmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later. << Less

  Curr. Microbiol. 62:1173-1178(2011) [PubMed] [EuropePMC]

• Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99-880 polygalacturonase gene family.

  Mertens J.A., Burdick R.C., Rooney A.P.

  A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% ... >> More

  A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with twelve genes encoding for endo-polygalacturonase enzymes and three genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygalacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times. << Less

  Fungal Genet. Biol. 45:1616-1624(2008) [PubMed] [EuropePMC]

• alpha-Galacturonidase(s): A new class of Family 4 glycoside hydrolases with strict specificity and a unique CHEV active site motif.

  Thompson J., Pikis A., Rich J., Hall B.G., Withers S.G.

  The catalytic activity of the Family 4 glycosidase LplD protein, whose active site motif is CHEV, is unknown despite its crystal structure having been determined in 2008. Here we identify that activity as being an α-galacturonidase whose natural substrate is probably α-1,4-di-galacturonate (GalUA2 ... >> More

  The catalytic activity of the Family 4 glycosidase LplD protein, whose active site motif is CHEV, is unknown despite its crystal structure having been determined in 2008. Here we identify that activity as being an α-galacturonidase whose natural substrate is probably α-1,4-di-galacturonate (GalUA2). Phylogenetic analysis shows that LplD belongs to a monophyletic clade of CHEV Family 4 enzymes, of which four other members are also shown to be galacturonidases. Family GH 4 enzymes catalyze the cleavage of the glycosidic bond, via a non-canonical redox-assisted mechanism that contrasts with Koshland's double-displacement mechanism. << Less

  FEBS Lett. 587:799-803(2013) [PubMed] [EuropePMC]

• Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen.

  Ibarrola I., Arilla M.C., Martinez A., Asturias J.A.

  <h4>Background</h4>Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens.<h4>Objective</h4>We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifo ... >> More

  <h4>Background</h4>Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens.<h4>Objective</h4>We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifolia.<h4>Methods</h4>P acerifolia pollen extract was fractionated by using ion-exchange and gel-permeation chromatography. Analyses were carried out by using ELISA, SDS-PAGE, isoelectrofocusing, and immunoblotting. Partial amino acid sequence was obtained by means of Edman sequencing of cyanogen bromide-digested peptides. Specific cDNA was cloned by using reverse transcription, followed by PCR, with amino acid sequences from peptides of the allergen.<h4>Results</h4>The allergen isolated from P acerifolia pollen, Pla a 2, is a glycoprotein with an observed molecular mass of 43 kd and an isoelectric point value of 9.3. It is involved in the allergic responses of 84% of patients with planetree-induced pollinosis and represented 52% of the total IgE-binding capacity of the P acerifolia extract. Pla a 2 displays polygalacturonase (PG) activity, being the first PG with functional enzyme activity from an angiosperm plant pollen described as an allergen. The cDNA allergen sequence codified for a 372-residue protein with 56% and 42% sequence identity to PGs from pollen and fruits, respectively. Western blot analysis showed that Pla a 2 is present in pollen and stems and has IgG cross-reactivity with a PG from tomato and pectate lyases from Cupressaceae pollen.<h4>Conclusion</h4>Pla a 2, a major allergen of P acerifolia pollen with PG activity has been purified, characterized, and cloned. << Less

  J. Allergy Clin. Immunol. 113:1185-1191(2004) [PubMed] [EuropePMC]