Analysis Of The ABC Transporter Family Gene Homologous Sequence Ctg16 Of Phanerochaete Chrysosporium

A set of ESTs relate to idophasic metabolism were obtained through suppression subtractive hybridization(SSH), cDNA microarray and DNA sequencing from the lignin degrading model fugus Phanerochaete chrysosporium grown under nitrogen-limited condition. Some of them are found to be homologous to ABC transporter family genes while BLASTX was carrying out against NCBI nr database. A 1023 bp sequence called ctgl6 was produced out of these ABC transporter homologs by contig assembly and proved to share great similarity with the nuclear binding domain (NBD) of ABC transporters.Gene prediction was carried out with a ctgl6 flanking context of 7250 bp on P. chrysosporium genome, from which a 6083 bp gene with a 3921 bp coding sequence and a putative peptide called Ctgl6pr of 1306 amino acids was deduced. When multiple-aligned with ctgl6 and the conferred CDS, the predicted gene was found to have exactly the same intron structure, comparing to the result of a ctgl6-genome sequence alignment. An additional NBD-like region was found in the 5'-rigion of ctgl6 on the predicted gene. Transmembrane domain prediction based on hydrophobicity analysis indicated a similar topology with that of a full ABC transporter, which further strengthened the conjecture that the gene represented by ctgl6 possesses a typical homodimer structure of normal ABC transporters.Three proteins of experimentally determined structure were found to have at least 30% identities with Ctgl6pr. The 3D model of Ctgl6pr was produced by the server, with a resolution of 3.1 A and root mean square (rms) of 0.75. A Procheck inspection of the model assigned it to a "better" state as 95% residues lies within the allowed regions of Ramachandran plot. Integrated examinations were performed andthe model was concluded to be accurate enough to carry out further structural and functional analysis. Most conserved domains shared by ABC transporters were on the proper position of the model. A proline to serine mutation on the Pro-loop that is essentially involved in communication between catalytic and signaling regions of NBD was found to have no impact on the overall structure of the loop. Analysis of dimerized model indicated that a ctgl6 protein dimmer should be involved in the hydrolyzation of ATP. The Gln68 on the opposite monomer may participate this process.