Crystal structure of the leucine aminopeptidase from Pseudomonas putida reveals the molecular basis for its enantioselectivity and broad substrate specificity.
The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and in complex with the inhibitor bestatin, show that the overall folding and hexameric organization of ppLAP are very similar to those of the closely related di-zinc leucine aminopeptidases (LAPs) from bovine lens and Escherichia coli. At pH 9.5, the active site contains two metal ions, one identified as Mn(2+) or Zn(2+) (site 1), and the other as Zn(2+) (site 2). By using a metal-dependent activity assay it was shown that site 1 in heterologously expressed ppLAP is occupied mainly by Mn(2+). Moreover, it was shown that Mn(2+) has a significant activation effect when bound to site 1 of ppLAP. At pH 5.2, the active site of ppLAP is highly disordered and the two metal ions are absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. A structural comparison of the ppLAP-bestatin complex with inhibitor-bound complexes of bovine lens LAP, along with substrate modelling, gave clear and new insights into its substrate specificity and high level of enantioselectivity.
Kale
Avinash
A
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Pijning
Tjaard
T
Sonke
Theo
T
Dijkstra
Bauke W
BW
Thunnissen
Andy-Mark W H
AM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D020536:Enzyme Activators / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000737:chemistry* / Q000378:metabolism*; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D016958:Pseudomonas putida / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008345:Manganese; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex; D015032:Zinc