Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron

Abstract

Some litter-decaying fungi secrete haem-thiolate peroxygenases that oxidize numerous organic compounds and therefore have a high potential for applications such as the detoxification of recalcitrant organic waste and chemical synthesis. Like P450 enzymes, they transfer oxygen functionalities to aromatic and aliphatic substrates. However, in contrast to this class of enzymes, they only require H(2)O(2) for activity. Furthermore, they exhibit halogenation activity, as in the well characterized fungal chloroperoxidase, and display ether-cleavage activity. The major form of a highly glycosylated peroxygenase was produced from Agrocybe aegerita culture media, purified to apparent SDS homogeneity and crystallized under three different pH conditions. One crystal form containing two molecules per asymmetric unit was solved at 2.2 A resolution by SAD using the anomalous signal of the haem iron. Subsequently, two other crystal forms with four molecules per asymmetric unit were determined at 2.3 and 2.6 A resolution by molecular replacement.

Figure 1

FPLC elution profile of the last step of purification on the strong cation exchanger Mono S. Inset: SDS–PAGE and isoelectric focusing PAGE of the pooled AaP fraction II. Lane 1, SDS–PAGE with molecular-weight markers (kDa); lane 2, SDS–PAGE of purified AaPII; lane 3, IEF gel of purified AaPII showing two isoforms; lane 4, IEF gel of pI markers.

Figure 2

Crystals of (a) AaPIIpH85, (b) AaPIIpH56 and (c) AaPIIpH46. The largest crystal of AaPIIpH85 has a length of 1.2 mm.

Figure 3

Diffraction patterns of (a) AaPIIpH85, (b) AaPIIpH56 and (c) AaPIIpH46 crystals. The edges of the frames correspond to 1.8, 2.2 and 2.3 Å, respectively.

Figure 4

Electron-density map at 2.52 Å resolution calculated after density modification in autoSHARP. The region of the map shows the haem (the iron is labelled) and its close environment at the proximal side comprising a helical segment containing the Fe-ligating cysteine.