Literature summary for 3.1.1.11 extracted from

Kent, L.M.; Loo, T.S.; Melton, L.D.; Mercadante, D.; Williams, M.A.; Jameson, G.B.
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 (2016), J. Biol. Chem., 291, 1289-1306 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene pmeB, DNA and amino acid sequence determination and analysis, recombinant expression in Pichia pastoris, the recombinant enzyme is secreted to the culture medium	Aspergillus niger

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme in deglycosylated and Asn-linked N-acetylglucosamine-stub forms, hanging drop vapor diffusion method, drops of 0.002 ml from protein and reservoir solutions in the ratios of 1:1 and 2:3, the latter containing 1.9 M (NH4)2SO4, 100 mM sodium acetate, pH 3.6, (EndoHf-deglycosylated Ani-PME2), or 1.8 M (NH4)2SO4, 100 mM sodium acetate, pH 4.1 (PNGaseF-deglycosylated Ani-PME2), 21°C, 1 week, method optimization, X-ray diffraction structure determination and analysis at 1.75-1.80 A resolution, molecular replacement using the structure of PMEs from Solanum lycopersicum (PDB code 1xg2) and Daucus carota (PDB code 1gq8) as search models	Aspergillus niger

Crystallization (Comment)

Organism

purified enzyme in deglycosylated and Asn-linked N-acetylglucosamine-stub forms, hanging drop vapor diffusion method, drops of 0.002 ml from protein and reservoir solutions in the ratios of 1:1 and 2:3, the latter containing 1.9 M (NH4)2SO4, 100 mM sodium acetate, pH 3.6, (EndoHf-deglycosylated Ani-PME2), or 1.8 M (NH4)2SO4, 100 mM sodium acetate, pH 4.1 (PNGaseF-deglycosylated Ani-PME2), 21°C, 1 week, method optimization, X-ray diffraction structure determination and analysis at 1.75-1.80 A resolution, molecular replacement using the structure of PMEs from Solanum lycopersicum (PDB code 1xg2) and Daucus carota (PDB code 1gq8) as search models

Aspergillus niger

Protein Variants

Protein Variants	Comment	Organism
additional information	for a Ani-PME2 construct, Saccharomyces cerevisiae consensus sequence AAAAAAATG and alpha-mating factor, as well as a Kex cleavage site AAAAGA are added to the sequence	Aspergillus niger

Metals/Ions

Metals/Ions	Comment	Organism	Structure
NaCl	required, at 0.8 M. Without added NaCl, protein activity is highly reduced. At pH 7.0 and above in the presence of 100 mM NaCl, Ani-PME2 is nearly inactive	Aspergillus niger

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
pectin + n H2O	Aspergillus niger	-	n methanol + pectate	-	?

Organism

Organism	UniProt	Comment	Textmining
Aspergillus niger	G3YAL0	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	Asn-linked N-acetylglucosamine-stub form enzyme, isozyme Ani-PME2 has two glycosylation sites. Uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate. Removal of N-linked high mannose glycans from Ani-PME is performed by MBP-tagged EndoHf or His-tagged PNGaseF, deglycosylated enzyme structure analysis, overview	Aspergillus niger

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Pichia pastoris cell-free culture medium by anion exchange chromatography and ultrafiltration	Aspergillus niger

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
homogalaturonan + H2O	-	Aspergillus niger	?	-	?
additional information	lack of processivity by isozyme Ani-PME2, overview	Aspergillus niger	?	-	?
pectin + n H2O	-	Aspergillus niger	n methanol + pectate	-	?
pectin + n H2O	capillary electrophoresis of de-methylesterified pectin, substrate is apple pectin with an approximately sample-averaged 35% degree of methylesterification. Non-processive (or near-random) de-methylesterification by Ani-PME2 at pH 4.2 or by strong base at pH 11.5 is confirmed by a plethora of fragments of varying length and charge. Unmodified apple pectin is preserved upon treatment with a strong base	Aspergillus niger	n methanol + pectate	-	?

Subunits

Subunits	Comment	Organism
More	structure analysis of the deglycosylated fungal isozyme Ani-PME2, which, while preserving key active-site residues, has distinctly different loop structures and surface electrostatic potential compared with plant, bacterial, and insect PMEs	Aspergillus niger

Synonyms

Synonyms	Comment	Organism
Ani-PME2	-	Aspergillus niger
pectin methylesterase	-	Aspergillus niger
PME	-	Aspergillus niger
pmeB	-	Aspergillus niger

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
35	-	assay at	Aspergillus niger

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.8	-	assay at	Aspergillus niger

pH Range

pH Minimum	pH Maximum	Comment	Organism
additional information	-	at pH 7.0 and above in the presence of 100 mM NaCl, Ani-PME2 is nearly inactive	Aspergillus niger

General Information

General Information	Comment	Organism
additional information	molecular dynamics simulations and electrostatic potential calculations. The substrate-binding groove is negatively charged. Enzyme sequence and activity comparisons to processive pectin methylesterases, e.g. from Aspergillus (Emericella) nidulans and Trichoderma reesei (Hypocrea jecorina), overview. Detailed structure analysis of a fungal isozyme Ani-PME2, which, while preserving key active-site residues, has distinctly different loop structures and surface electrostatic potential compared with plant, bacterial, and insect PMEs, molecular dynamics simulations on Ani-PME2. Homology modeling of the structure of isozyme Ani-PME1	Aspergillus niger
physiological function	Aspergillus niger contains a non-processive, salt-requiring, and acidophilic pectin methylesterase	Aspergillus niger