Biochemical and protein structural analyses of XveII mutant enzymes that recognize extended SmaI cognate sequence

• Main Authors: Yu-Jen Yu, 于玉珍
• Other Authors: 楊明德
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/96719642694977314758

博士 === 中興大學 === 分子生物學研究所 === 95 === The XveII restriction-modification (R-M) system, xveIIM and xveIIR, is a SmaI-like R-M system and cloned from Xanthomonas campestris pv. vesicatoria strain 7-1 (Xcv7-1). The xveIIM encoding the XveII methyltransferase (M.XveII) methylates the second cytosine of the 5′-CCCGGG-3′ recognition sequence and belongs to the m4C-methyltransferase family. However, an internal TAA stop codon was found to locate at nucleotide 484 – 486 (the 162nd amino acid residue) of the predicted xveIIR ORF resulting in an 18.3 kDa nonfunctional protein product. Site-directed mutagenesis was applied to replace the internal TAA stop codon of the xveIIR gene with twenty different amino acid codons and designated as xveIIR-mut. Each of the recombinant XveII-mut was overexpressed and purified from E. coli ER2566 and most of the 31 kDa XveII mutant enzymes restore restriction activities. The specificities of XveII mutant enzymes, being identical to SmaI, cleave between C and G of the 5′-CCCGGG recognition sequence and yield a blunt-ended product. The cleavage activity of the tryptophan substituted enzyme (XveII-W) is the highest among the mutants, however, still lower than that of SmaI. The mutant XveII-S was found to have the highest binding affinity to the cognate DNA (KD = 97.4 nM). According to the results of glutaraldehyde cross-linking experiments and size exclusion chromatography, XveII-mut was determined to exist as a dimer. XveII-mutD was constructed by ligating two xveIIR-mut genes with 4 continuous glycine codons and expressed as a single polypeptide dimer of XveII-mut with 63 kDa molecular weight. The specificities of XveII-mutD were the same as those of the XveII-muts while the DNA cleavage, binding activities, and stability of XveII-mutD were higher than those of the corresponding XveII-mut. The mutant enzymes exhibited different activities, indicating that different amino acid substitutions at the 162nd position might play an important role in XveII. Results showed that the cleavage rate and the DNA binding activites of XveII-mut were influenced by different flanking sequences of the recognition site. Plasmid and linealized DNA with different flanking sequence of the recognition site were used for digestion and revealed that XveII-WD prefer A or T immediately adjacent to the SmaI sites. Base composition next to the CC flanking sequence of the SmaI site had influence on the cleavage rate. Moreover, the one with GC flanking sequence had the lowest cleavage rate. To explore the roles of the 162nd amino acid in XveII-mut the crystal structure of the DNA-free XveII-I was determined and analyzed at 2.7Å resolution. Results showed that structure of XveII-I spatial superimposed with the Endo domain of NaeI and suggested that E80-YD105-K116 is the active site motif and Asp241, Glu245 and Lys247 are involved in DNA binding. Alanine-substitution at these predicted amino acid residues were constructed and found that these XveII-I mutants lost cleavage activity on double-stranded DNA. In addition, mutant XveIIK247A was almost completely lost DNA binding activity. Amino acid alignment revealed that E72-HD98-K109 are the catalytic residues of SmaI and both of XveII and SmaI are not typical PD-(D/E)XK superfamily nucleases. Based on the crystal structure of XveII-I, we proposed that the three hydrophobic residues, Ile21 and Val24 and Phe69, are responsible for its dimerization by hydrophobic contacts between helix bundles in its N-terminal region. The 162nd amino acid residue of XveII is located between α4 and α5 of the α/β core structure near the metal ion binding region. The differences in cleavage activities of the XveII-mut may due to comformational change when variants of the XveII-mut binding with substrate DNA.