PPLO is a copper-containing amine oxidase from the yeast P. pastoris. In contrast to the structurally characterized CAOs, PPLO has similar substrate specificity and inhibitor sensitivity to the mammalian enzyme. Further, it is 30% identical to the human kidney diamine oxidase (the highest of any non- mammalian source). While both PPLO and the mammalian lysyl oxidase oxidize peptidyl lysine residues, PPLO is much larger (a dimer of ≈150 kDa compared to a monomer of ≈30 kDa) and employs the cofactor TPQ: rather than LTQ: As structural studies on mammalian lysyl oxidase have been seriously limited by its marked insolubility and aggregation tendency, it was hoped that determining the structure of PPLO would help elucidate the molecular basis for the recognition and oxidative deamination of peptidyl lysine residues. Pichia pastoris Lysyl Oxidase (PPLO) This work is based upon research conducted at the Stanford Synchrotron Radiation Laboratory (SSRL). SSRL is funded by the Department of Energy, Office of Basic Energy Sciences. The Biotechnology Program is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program and the Department of Energy, Office of Biological and Environmental Research. Paul Ellis, Aina Cohen: Stanford Synchrotron Radiation Laboratory Anthony Duff, Mihwa Lee, Katrina Willingham, David Langley, Megan Maher, Hans Freeman, J. Mitchell Guss: University of Sydney Jason Kuchar, David Dooley: Montana State University PPLO Copper amine oxidases Copper amine oxidases (CAOs) are ubiquitous metalloenzymes catalyzing the oxidative deamination of amines to aldehydes and ammonia: RCH 2 NH 2 + O 2 + H 2 O  RCHO + NH 3 + H 2 O 2 Due to their typically broad substrate specificities it has sometimes proven difficult to assign a specific role for amine oxidases but it is known that in vertebrates the enzyme lysyl oxidase is responsible for the oxidation of selected lysine residues in collagen and elastin precursors, a step critical for the maturation of connective tissues. The enzyme may also be involved in the regulation of polyamines, and has been implicated in tumor suppression. Initial maps were poor (R=0.59) Improvements were obtained by: Deleting ~100 residues Arp/warp NCS averaging Simulated annealing Current model contains: 4 × 734 ordered residues (2 dimers) 4 × 5 carbohydrate attachments 4 active site Cu 2+ ions 8 Ca 2+ ions 3988 ordered H 2 O 28 SO 4 2- ions R=0.15, R free =0.18 RefinementStructure solution Data was collected at SSRL beamline 9-1 unit cell: C2: × × Å, β=124.8  Å, reflections, R merge =0.06 The structure was solved by Anthony Duff using molecular replacement. The search model was selected from the PDB database using Blast: E. coli CAO (1OAC), A/B chains, residues The PPLO active site lies at the base of a large funnel: What’s different? 15 Å wide at 12 Å above the TPQ 10 Å wide at 3 Å above the TPQ Peak height above the TPQ is 24 Å HPAO Only a few Å wide, but 14 Å deep All previous CAOs have a narrow entrance channel: and even bulky substrates like a beta finger can access the active site: Cutaway view Questions: - What is the “native” substrate of PPLO? - Is PPLO a good model for a “true” lysyl oxidase? The structure is similar to other dimeric CAOs: The structure of PPLO PPLO HPAO AGAO PSAO ECAO TPQ in “Cu OFF” or “active” state TPQ in “Cu ON” or “inactive” state ASP 98, the putative active-site base and has a typical copper amine oxidase active site: at the base of the usual deep depression: