title: Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control.
abstract: Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methyl ester groups of the homogalacturonan component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively, describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high resolution x-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10⅔-turn parallel β-helix (similar to but with less extensive loops than bacterial, plant, and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic, and acidophilic. Molecular dynamics simulations and electrostatic potential calculations reveal very different behavior and properties compared with processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged.
authors.affiliation: From Riddet Institute and Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Kent
authors.affiliation: From Riddet Institute and Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
authors.forename: Trevor S
authors.initials: TS
authors.lastname: Loo
authors.affiliation: From Riddet Institute and School of Chemical Sciences, University of Auckland, Auckland 1142, New Zealand.
authors.forename: Laurence D
authors.initials: LD
authors.lastname: Melton
authors.affiliation: From Riddet Institute and Molecular Biomechanics Group, Heidelberg Institute for Theoretical Studies, Schloss-Wolfsbrunnenweg, 69118 Heidelberg, Germany, and.
authors.forename: Davide
authors.initials: D
authors.lastname: Mercadante
authors.affiliation: From Riddet Institute and Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand, MacDiarmid Institute for Advanced Materials and Nanotechnology, Palmerston North 4442, New Zealand m.williams@massey.ac.nz.
authors.forename: Martin A K
authors.initials: MA
authors.lastname: Williams
authors.affiliation: From Riddet Institute and Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand, MacDiarmid Institute for Advanced Materials and Nanotechnology, Palmerston North 4442, New Zealand g.b.jameson@massey.ac.nz.
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Jameson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D016384:Consensus Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D005656:Fungal Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase
keywords: capillary electrophoresis; carbohydrate processing; crystal structure; electrostatics; molecular dynamics; pectin methylesterase; processivity