The T. cervina LiP genomic gene tclipG was successfully cloned using the primers tclipg-S and tclipg-A designed from 5′- and 3′-untranslated regions of the tclip sequence. tclipG (GenBank accession no. AB237774) contains a 2047-bp translated region ending with a TAA termination codon, and contains 17 introns and 18 exons (Fig. S2). All splicing junction sequences of introns strictly adhere to the GT-AG rule. Lariat consensus sequences (5′-NNHTNAY-3′) were also found in all intronic sequences. The exons exhibited a high
G+C content (59.6%), while introns had a much lower G+C content (42.6%). selleck inhibitor Although the G+C content is lower than those of other LiP genes (Gold & Alic, 1993), it is consistent with that of a recently reported VP gene from Bjerkandera (Moreira et al., 2005). The lengths of introns were almost constant (about 50 bp), while the lengths of exons were much more diverse (5–240 bp). Short exons encoding <10 amino acids (named micro-exons) have also been found in cytochrome P450 genes from P. chrysosporium and are considered to be a specific feature of fungal P450 genes and to be related to the diversity of these genes (Doddapaneni et al., 2005). The schematic representations of 15 fungal peroxidase genes are shown in Fig. 4. RG7420 The T. cervina LiP intron/exon structure is relatively
similar to that of Coprinopsis cinereus peroxidase (CIP), which does not show ligninolytic activity, and is rather different from those of other ligninolytic peroxidases. To further clarify the evolutionary attributes of T. cervina LiP, we constructed a phylogenetic tree with 15 fungal peroxidases Janus kinase (JAK) that have been
characterized enzymatically (Fig. 5). LiP, VP, and manganese peroxidases form their own clusters based on fungal species and catalytic types. However, T. cervina LiP was not classified into any of these clusters, even though T. cervina LiP is a LiP-type catalyst. These results suggested that T. cervina LiP is evolutionarily distant from LiP and VP, despite the fact that T. cervina LiP is functionally a LiP-type peroxidase. We identified the cDNA and genomic DNA encoding T. cervina LiP and characterized the T. cervina LiP molecule. Comparison of LiP sequences revealed that the T. cervina LiP sequence lacks the tryptophan corresponding to Trp171 of P. chrysosporium LiP, but contains a unique Tyr181. Structural analysis using a homology model provided evidence that Tyr181 plays a role in the electron transfer part of the T. cervina LiP catalytic mechanism and is probably the substrate-oxidation site, although further structural and kinetic studies are required to confirm this. Evolutionary analyses indicated that T. cervina LiP does not share the same origin as LiP, suggesting that T. cervina LiP has acquired LiP-type catalytic properties via convergent evolution. Thus, we concluded that T. cervina LiP could be a novel fungal peroxidase with a new LiP-catalytic mechanism including Tyr181. Fig. S1.