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

Abstract

XveII 限制修飾系統是由 Xanthomonas campestris pv. vesicatoria 7-1 (Xcv7-1) 菌株中所選殖出來，包含 xveIIM 及 xveIIR 基因，為一種 SmaI-like 限制修飾系統。xveIIM 基因可轉譯出 XveII 甲基轉移酶 (M.XveII)，會將 5′-CCCGGG-3′ 中第二個 C 進行甲基化，是屬於 m4C-甲基轉移酶。而 xveIIR 基因預測為一含 813 鹼基對之 ORF，但在第 484 至 486 個鹼基對 (即第 162 個胺基酸位置) 具有一內在 TAA 終止密碼，其表現出來的 XveII 分子量僅 18.3 kDa 為一無功能蛋白。為探討第 162 胺基酸位置在 XveII 所扮演的角色，本研究針對 xveIIR 基因，以定點突變的方法將此基因內在 TAA 終止密碼以二十種不同胺基酸密碼子取代，並命名為 xveIIR-mut 基因。突變基因於 E. coli ER2566 菌株表現與純化後，可得到約 31 kDa 的 XveII-mut，並發現大部份取代株可以恢復限制酶活性，切割在5′-CCCGGG-3′辨識序列的 C 與 G 之間形成平端的產物 (CCC↓GGG)，且切割活性以 tryptophan 取代株 (XveII-W) 最高，但卻遠低於 SmaI 的切割活性；而與 DNA 的親和力則是以 XveII-S 為最佳 (KD = 97.4 nM)。藉由交聯反應與分子篩管柱分析，XveII-mut 是以雙隅體的構形呈現。因此亦利用四個連續 glycine 的密碼子 (GGC) 將二個相同之 xveIIR-mut 基因串聯，可以表現出由 555 個胺基酸所構成之單一胜肽雙隅體 (XveII-mutD)。所表現之 XveII-mutD 並未改變其專一性，且其切割活性與 DNA 結合能力及穩定性等皆優於 XveII-mut。在第 162 位置由不同胺基酸取代株，會造成活性之差異，顯示此位置之胺基酸在 XveII 中可能扮演重要角色。根據 DNA 切割的結果分析，XveII-mut 對於帶有不同鄰邊序列的辨識切位，具有不同的切割效率與結合能力。利用帶有不同鄰邊序列的質體或線形 DNA 的切割實驗，顯示 XveII-WD 較偏好 A 或 T 之相鄰序列；至於 CC 的組合要視更外側的核苷酸序列才可決定其切割速率；對於 GC 鄰邊序列切割速率則最慢。進一步藉由 XveII-I 結構分析，了解 XveII 中第 162 胺基酸位置可能扮演之角色，同時推測 SmaI 中重要胺基酸位置。由 XveII-I 結晶繞射得到 2.7Å 的解析度，經比對與 NaeI 限制酶的 Endo domain 具有空間上的重疊性，推測活性中心區域為 E80-YD105-K116，而參與 DNA 結合的胺基酸為 Asp241、Glu245 與 Lys247，將這些位置以 alanine 取代後皆喪失切割能力，其中 XveII-IK247A 則幾乎喪失了 DNA 結合能力。藉由胺基酸序列的比對，推測 SmaI 的活性中心胺基酸為 E72-HD98-K109，與 XveII-I 的活性中心皆為非典型的 PD-(D/E)XK superfamily。XveII-I 形成雙隅體介面是位在 N-端區域 helix bundles 間形成疏水性作用力，即由胺基酸 Ile21、Val24和 Phe69 所形成。至於第 162 胺基酸位置是座落於 α/β 結構中心的 α4 與 α5 之間，接近金屬離子結合區域，因此推測 XveII 第 162 位置由不同胺基酸的取代，會改變與 DNA 之間構形改變，進而導致活性的差異性。

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.