CONSERVED SEQUENCE MOTIF DPPY IN REGION-IV OF THE PHAGE-T4 DAM DNA-[N6-ADENINE]-METHYLTRANSFERASE IS IMPORTANT FOR S-ADENOSYL-L-METHIONINE BINDING
CONSERVED SEQUENCE MOTIF DPPY IN REGION-IV OF THE PHAGE-T4 DAM DNA-[N6-ADENINE]-METHYLTRANSFERASE IS IMPORTANT FOR S-ADENOSYL-L-METHIONINE BINDING
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DOI：
10.1093/nar/21.20.4659
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发表时间：
1993-10-11
影响因子：
14.9
通讯作者：
HATTMAN, S
中科院分区：
文献类型：
--
作者：
KOSSYKH, VG;SCHLAGMAN, SL;HATTMAN, S
对DNA-[N6-腺嘌呤]-甲基转移酶的氨基酸序列进行比较，发现了几个保守区。所有这些酶都含有一个DPPY[或密切相关的]基序。通过对克隆的T4Dam基因进行定点突变，我们将该基序中的第一个Pro残基[位于T4Dam-MTase的保守区域IV]改变为丙氨酸或苏氨酸。对突变型P172A和P172T进行了高效生产和纯化。动力学研究表明，与野生型[wt]相比，两种突变形式具有：(I)底物S-腺苷蛋氨酸[ADOMet]的K(M)分别增加[5倍和20倍]；(Ii)k(Cat)略有降低[2和4倍]；(Iii)k(Cat)/K(M)ADOMet[10和100倍]明显降低；以及(Iv)底物DNA的K(M)几乎相同。平衡透析研究表明，突变酶对Adobe Met的K(A)降低了[4倍和9倍]。综上所述，这些数据表明，P172A和P172T的改变主要导致了对Adobe Met的亲和力降低。这表明DPPY基序对于Adobe Met结合是重要的，并且区域IV包含或是Adobe Met结合位点的一部分。
Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransterases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] K(m) for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] k(cat); (iii) a strongly reduced k(cat)/K(m)AdoMet [10 and 100-fold]; and (iv) almost the same K(m) for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] K(a) for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.