Stachelhaus T, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Biochemistry. 2000 May 16;39(19):5775-87. doi: 10.1021/bi9929002.
The epimerase (E) domain of the three-domain (ATE) initiation module of Bacillus brevis gramicidin S synthetase equilibrates the Calpha configuration of the phenylalanyl moiety presented as Phe-S-4'-phosphopantetheine-modified (Ppant) acyl enzyme. Mutants at 22 residues of this E domain that are conserved across the approximately 450 residue E domains of nonribosomal peptide synthetases were constructed, and the PheATE derivatives expressed in Escherichia coli as C-terminal His tag fusions and then purified and assayed for three activities: (1) the L-Phe Calpha-[(3)H] exchange to solvent, (2) the rate of approach to D-Phe/L-Phe-S-Ppant acyl enzyme equilibrium from either L- or D-Phe, and (3) the rate of Phe-Pro dipeptidyl-S-Ppant enzyme formation with the downstream ProCAT module. We found that for wild-type PheATE epimerization is much faster than subsequent condensation, leading to a 1.9:1 ratio of D-Phe-S-Ppant/L-Phe-S-Ppant acyl enzyme. Only D-Phe is then transferred to yield D-Phe-L-Pro-S-Ppant ProCAT acyl enzyme. Among the mutants generated, three PheATE constructs, H753A, D757S, and Y976A, showed no detectable Calpha-(3)H washout, while E892A and R896A were among a larger set partially impaired. All these mutants were dramatically impaired in approach to D-Phe/L-Phe-S-Ppant equilibrium from either D- or L-Phe, while another construct, D767S, was asymmetrically impaired only for D-to-L-Phe direction. In the D-Phe-L-Pro dipeptidyl-S-Ppant condensation assay, the H753A and E892A forms of PheATE were only slightly active from L-Phe but unimpaired from D-Phe; N975A epimerizes faster than Y976A from L-Phe. When the chirality of the Phe-Pro-diketopiperazine released product was analyzed the D,L/L,L ratio from wild-type PheATE and ProCAT was 98:2. From E892A and N975A it was comparably 95:5 and 92:8, but H753A and Y976A yielded 56% of the L,L-product, reflecting a gain of function to transfer L-Phe. The 98:2 preference of wild-type PheATE for D-Phe transfer reflects the kinetically controlled stereopreference of the condensation (C) domain of ProCAT for the D-Phe-S-Ppant donor substrate. It may be that other NRPS C domains immediately downstream of E domains will likewise be D-selective.
短短芽孢杆菌短杆菌肽S合成酶的三结构域（ATE）起始模块中的差向异构酶（E）结构域，可平衡以苯丙氨酰基部分呈现为苯丙氨酰 - S - 4'-磷酸泛酰巯基乙胺修饰（Ppant）的酰基酶的Cα构型。构建了该E结构域中22个残基的突变体，这些残基在非核糖体肽合成酶约450个残基的E结构域中是保守的。在大肠杆菌中表达的苯丙氨酰 - ATE衍生物作为C末端His标签融合蛋白，然后进行纯化，并检测三种活性：（1）L - 苯丙氨酸Cα - [³H]与溶剂的交换；（2）从L - 或D - 苯丙氨酸达到D - 苯丙氨酸/L - 苯丙氨酸 - S - Ppant酰基酶平衡的速率；（3）与下游脯氨酸缩合酶（ProCAT）模块形成苯丙氨酰 - 脯氨酸二肽基 - S - Ppant酶的速率。我们发现，对于野生型苯丙氨酰 - ATE，差向异构化比随后的缩合快得多，导致D - 苯丙氨酸 - S - Ppant/L - 苯丙氨酸 - S - Ppant酰基酶的比例为1.9:¹。然后仅转移D - 苯丙氨酸以产生D - 苯丙氨酰 - L - 脯氨酸 - S - Ppant ProCAT酰基酶。在产生的突变体中，三种苯丙氨酰 - ATE构建体H753A、D757S和Y976A未检测到可检测到的Cα - ³H洗脱，而E892A和R896A属于部分受损的较大一组。所有这些突变体从D - 或L - 苯丙氨酸达到D - 苯丙氨酸/L - 苯丙氨酸 - S - Ppant平衡的过程中均受到显著损害，而另一个构建体D767S仅在D - 到L - 苯丙氨酸方向上不对称受损。在D - 苯丙氨酰 - L - 脯氨酸二肽基 - S - Ppant缩合试验中，苯丙氨酰 - ATE的H753A和E892A形式从L - 苯丙氨酸仅有轻微活性，但从D - 苯丙氨酸未受损；N975A从L - 苯丙氨酸的差向异构化比Y976A快。当分析释放产物苯丙氨酰 - 脯氨酸 - 二酮哌嗪的手性时，野生型苯丙氨酰 - ATE和ProCAT的D,L/L,L比例为98:²。来自E892A和N975A的比例分别为95:⁵和92:⁸，但H753A和Y976A产生了56%的L,L - 产物，反映了转移L - 苯丙氨酸的功能获得。野生型苯丙氨酰 - ATE对D - 苯丙氨酸转移的98:²偏好反映了ProCAT缩合（C）结构域对D - 苯丙氨酸 - S - Ppant供体底物的动力学控制立体偏好。可能在E结构域下游紧邻的其他非核糖体肽合成酶C结构域同样会是D - 选择性的。
