Trauger J W, Kohli R M, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Biochemistry. 2001 Jun 19;40(24):7092-8. doi: 10.1021/bi010035r.
The excised C-terminal thioesterase (TE) domain from the multidomain tyrocidine nonribosomal peptide synthetase (NRPS) was recently shown to catalyze head-to-tail cyclization of a decapeptide thioester to form the cyclic decapeptide antibiotic tyrocidine A [Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215-218]. The peptide thioester substrate was a mimic of the TE domain's natural, synthetase-bound substrate. We report here the synthesis of modified peptide thioester substrates in which parts of the peptide backbone are altered either by the replacement of three amino acid blocks with a flexible spacer or by replacement of individual amide bonds with ester bonds. Rates of TE domain catalyzed cyclization were determined for these substrates and compared with that of the wild-type substrate, revealing that some parts of the peptide backbone are important for cyclization, while other parts can be modified without significantly affecting the cyclization rate. We also report the synthesis of a modified substrate in which the N-terminal amino group of the wild-type substrate, which is the nucleophile in the cyclization reaction, is replaced with a hydroxyl group and show that this compound is cyclized by the TE domain to form a macrolactone at a rate comparable to that of the wild-type substrate. These results demonstrate that the TE domain from the tyrocidine NRPS can catalyze cyclization of depsipeptides and other backbone-substituted peptides and suggest that during the cyclization reaction the peptide substrate is preorganized for cyclization in the enzyme active site in part by intramolecular backbone hydrogen bonds analogous to those in the product tyrocidine A.
最近研究表明，从多结构域短杆菌酪肽非核糖体肽合成酶（NRPS）中切除的C末端硫酯酶（TE）结构域可催化十肽硫酯的头对尾环化反应，形成环状十肽抗生素短杆菌酪肽A [Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., and Walsh, C. T. (2000) Nature 407, 215 - 218]。肽硫酯底物是TE结构域天然的、与合成酶结合的底物的模拟物。我们在此报告了修饰肽硫酯底物的合成，其中肽主链的部分结构通过用柔性间隔物替换三个氨基酸块或用酯键替换单个酰胺键而发生改变。测定了这些底物在TE结构域催化下的环化速率，并与野生型底物的环化速率进行比较，结果表明肽主链的某些部分对环化很重要，而其他部分可以被修饰而不会显著影响环化速率。我们还报告了一种修饰底物的合成，其中野生型底物的N末端氨基（环化反应中的亲核试剂）被羟基取代，并表明该化合物被TE结构域环化形成大环内酯，其速率与野生型底物相当。这些结果表明，短杆菌酪肽NRPS的TE结构域可以催化缩肽和其他主链取代肽的环化反应，并表明在环化反应过程中，肽底物在酶活性位点通过分子内主链氢键进行预环化，类似于产物短杆菌酪肽A中的氢键。
