The Structure of the Lysyl Oxidase from Pichia pastoris - a Copper-containing Amine Oxidase that can Oxidise Lysine-containing Peptides

Anthony P. Duff,^a Paul J. Ellis,^b Aina E. Cohen,^b Jason A. Kuchar,^c David Langley,^a David M. Dooley,^c Hans C. Freeman,^a and J. Mitchell Guss^a

^aSchool of Molecular and Microbial Biosciences, The University of Sydney, Sydney Australia; ^bThe Stanford Synchrotron Research Laboratory, Menlo Park, California, USA; ^cDepartment of Chemistry & Biochemistry, Montana State University, Bozeman, Montana, USA (A.Duff@usyd.edu.au)

We have determined the structure of the lysyl oxidase from Pichia pastoris (PPLO) at 1.65Å resolution with R=16.0% and R_Free=18.7%. Crystals belong to space group C2, with a=249Å b=120Å c=152Å and b=125º [1]. The asymmetric unit contains two dimers, thus providing four-fold non-crystallographic symmetry. The structure was solved by molecular replacement, despite the search model having only a 25% identity match to 66% of the sequence.

PPLO is a copper amine oxidase (CuAO) [2]. CuAOs catalyse the oxidative deamination of a primary amine to produce an aldehyde, ammonia and hydrogen peroxide (R‑CH₂‑NH₂ + O₂ + H₂O à R‑CHO + NH₃ + HOOH). They contain Cu(II) and utilise a side chain derived cofactor that is autocatalytically generated in the presence of Cu(II) and O₂.

PPLO is the fifth CuAO to be structurally characterised, after the CuAOs from the bacteria Escherichia coli and Arthrobacter globiformis, the yeast Hansenula polymorpha, and a higher plant Pisum sativum. It is like the previous four in overall architecture, but has some substantially different features consistent with its unique ability to oxidise the side chains of lysine residues in peptides - the initial step in the reactions for the cross-linking of collagen and elastin. This functional activity (in vitro) relates PPLO to another class of CuAOs, having a different size, sequence and cofactor, known as the lysyl oxidases. No bona fide lysyl oxidases have been structurally characterised to date.

The structure of PPLO explains its unusual substrate specificity. Other CuAOs can oxidase only small organic amines, such as methylamine or benzylamine, but are unable to oxidise lysyl peptides since they have buried active sites and narrow substrate channels. PPLO on the other hand has a broad open funnel leading from the solvent to the active-site cofactor. We have modelled putative peptide substrates into this site.

A solvent lake lies between the otherwise tightly associated subunits in the dimers. This lake is larger in PPLO and borders the ligands of the active site copper ion. This supports the proposal that the smaller reactants and products (O₂, H₂O, NH₃, HOOH and H⁺) may enter or leave the active site via this lake.

References

1 Lee M, Willingham K, Langley D, Maher MJ, Cohen AE, Ellis PJ, Kuchar JA, Dooley DM, Freeman HC, Guss JM. (2002) Acta Crystallogr D. 58, 2177-9 “Crystallization of Pichia pastoris lysyl oxidase”

2 Dove JE, Smith AJ, Kuchar J, Brown DE, Dooley DM, Klinman JP.(1996) FEBS Lett. 398, 231-4. “Identification of the quinone cofactor in a lysyl oxidase from Pichia pastoris”