Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana

Abstract

Zearalenone (ZEN) is a mycotoxin which causes huge economic losses in the food and animal feed industries. The lactonase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, is the only known ZEN-detoxifying enzyme. Here, a protein homologous to ZHD101, denoted CbZHD, from Cladophialophora batiana was expressed and characterized. Sequence alignment indicates that CbZHD possesses the same catalytic triad and ZEN-interacting residues as found in ZHD101. CbZHD exhibits optimal enzyme activity at 35°C and pH 8, and is sensitive to heat treatment. The crystal structure of apo CbZHD was determined to 1.75 Å resolution. The active-site compositions of CbZHD and ZHD101 were analyzed.

Keywords: Cladophialophora bantiana; crystal structure; lactonases; mycotoxins; zearalenone.

Figure 1

Sequence alignment of ZHD101 and CbZHD. This figure was produced using ESPript (Gouet et al., 2003 ▸). Catalytic triads and substrate-interacting residues are labelled with pink and blue dots, respectively.

Figure 2

The effect of pH/temperature and the thermostability of CbZHD. (a) The ZEN-hydrolytic activity of recombinant CbZHD was measured at various pH values (left) and temperatures (right). The relative activity of each sample is presented as a percentage of the maximal value. (b) CbZHD was treated at various temperatures for 1, 2 or 10 min prior to activity measurement. The relative activity of each sample is presented as a percentage of that of the untreated sample.

Figure 3

Overall structure and catalytic mechanism of CbZHD. (a) The four polypeptide chains in the asymmetric unit of a CbZHD crystal. The upper and lower pairs of monomers form two dimers. (b) The overall structure of CbZHD is shown as a cartoon model. The ZEN was modelled from the structure of the complex of an inactive form of ZHD101 with ZEN (PDB entry 3wzm; Peng et al., 2014 ▸) and is shown in stick representation. (c) Superimposition of CbZHD and ZHD101. ZEN from the structure of its complex with inactive ZHD101 was modelled and the residues surrounding ZEN in CbZHD and ZHD101 are shown in green and cyan, respectively. The catalytic triad residues are shown in purple/blue. Dashed lines indicate distances of <2.0 Å. (d) The proposed catalytic mechanism of CbZHD. The attacking water molecule is shown in blue.