title: Biochemical characteristics of an alkaline pectate lyase PelA from Volvariella volvacea: roles of the highly conserved N-glycosylation site in its secretion and activity.
abstract: Alkaline pectate lyases have great application potential in the bioscouring of textiles. They are isolated predominantly from bacteria and a few fungi. Here, we report the biochemical characteristics of a novel alkaline pectate lyase PelA from the basidiomycete Volvariella volvacea. The full-length pelA encodes a 321-amino-acid polypeptide containing a putative 18-residue signal peptide and a pectate lyase family 1 catalytic domain. It contains one conserved and one non-conserved potential N-glycosylation site (N-X-S/T) at the residues N95 and N198, respectively. The enzyme showed optimal activity at 60 °C and pH 10, although it was stable between pH 4 and pH 11. Additional Ca(2+) was not required to measure PelA activity in vitro, but it could significantly enhance its activity and thermal stability. The V max values using polygalacturonic acid as substrate were increased from 50.71 to 89.96 IU mg(-1) by the addition of 0.1 mM Ca(2+), whereas the K m values were decreased from 0.681 to 0.514 mg ml(-1). Site-directed mutagenesis revealed PelA has only one N-glycan attached to the residue N95. This N-glycan is crucial to its efficient secretion and activity possibly due to its role in maintaining the secondary structure of PelA. Amino acid substitution at the residue N198 had no effect on PelA secretion, but resulted in a slight (5.16 %) to modest (27.37 %) decrease in specific activity and less thermal stability, indicating the amino acid itself is also important for activity due to it being highly conserved and because of its proximity to the catalytic site.
authors.affiliation: Department of Biological Engineering, College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.
authors.forename: Aiqin
authors.initials: A
authors.lastname: Shi
authors.forename: Hang
authors.initials: H
authors.lastname: Hu
authors.forename: Fei
authors.initials: F
authors.lastname: Zheng
authors.forename: Liangkun
authors.initials: L
authors.lastname: Long
authors.forename: Shaojun
authors.initials: S
authors.lastname: Ding
mesh_terms: D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D021382:Protein Sorting Signals; D017422:Sequence Analysis, DNA; D013696:Temperature; D055367:Volvariella / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D020536:Enzyme Activators; D021382:Protein Sorting Signals; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D002118:Calcium; C003181:polygalacturonic acid