title: Structural investigations of the active-site mutant Asn156Ala of outer membrane phospholipase A: function of the Asn-His interaction in the catalytic triad.
abstract: Outer membrane phospholipase A (OMPLA) from Escherichia coli is an integral-membrane enzyme with a unique His-Ser-Asn catalytic triad. In serine proteases and serine esterases usually an Asp occurs in the catalytic triad; its role has been the subject of much debate. Here the role of the uncharged asparagine in the active site of OMPLA is investigated by structural characterization of the Asn156Ala mutant. Asparagine 156 is not involved in maintaining the overall active-site configuration and does not contribute significantly to the thermal stability of OMPLA. The active-site histidine retains an active conformation in the mutant notwithstanding the loss of the hydrogen bond to the asparagine side chain. Instead, stabilization of the correct tautomeric form of the histidine can account for the observed decrease in activity of the Asn156Ala mutant.
authors.affiliation: Laboratory of Biophysical Chemistry, BIOSON Research Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
authors.forename: H J
authors.initials: HJ
authors.lastname: Snijder
authors.forename: J H
authors.initials: JH
authors.lastname: Van Eerde
authors.forename: R L
authors.initials: RL
authors.lastname: Kingma
authors.forename: K H
authors.initials: KH
authors.lastname: Kalk
authors.forename: N
authors.initials: N
authors.lastname: Dekker
authors.forename: M R
authors.initials: MR
authors.lastname: Egmond
authors.forename: B W
authors.initials: BW
authors.lastname: Dijkstra
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054466:Phospholipases A1; D011487:Protein Conformation; D011489:Protein Denaturation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D006639:Histidine; D001216:Asparagine; D010741:Phospholipases A; D054466:Phospholipases A1; C412876:outer membrane phospholipase A; D000409:Alanine