Liu S, Yahr T L, Frank D W, Barbieri J T
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
J Bacteriol. 1997 Mar;179(5):1609-13. doi: 10.1128/jb.179.5.1609-1613.1997.
Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.
遗传学研究表明，铜绿假单胞菌外切酶S的53-kDa（Exo53）和49-kDa（ExoS）形式分别由单独的基因编码，分别称为exoT和exoS。尽管ExoS和Exo53具有76%的一级氨基酸同源性，但已证明Exo53的ADP-核糖基转移酶活性约为ExoS比活性的0.2%。通过使用一种重组缺失蛋白分析了Exo53相对于ExoS较低的ADP-核糖基转移酶活性的机制，该重组缺失蛋白包含Exo53的催化结构域，由其223个羧基末端残基组成（称为N223-53）。N223-53在大肠杆菌中表达为一种稳定的可溶性融合蛋白，纯化后纯度>80%。在线性速度条件下，N223-53催化大豆胰蛋白酶抑制剂（SBTI）的FAS（外切酶S激活因子）依赖性ADP-核糖基化的速率为N222-49催化速率的0.4%，催化Ras蛋白的ADP-核糖基化的速率为N222-49催化速率的1.0%。N222-49是一种由ExoS的222个羧基末端残基组成的蛋白质，代表其催化结构域。N223-53对NAD和SBTI的结合亲和力与N222-49相似（Km值差异小于五倍），但对SBTI的ADP-核糖基化显示出较低的速度。这表明Exo53进行ADP-核糖基化的主要缺陷在于其催化能力。对由N223-53和N222-49的相互对应部分组成的杂合蛋白的分析将催化缺陷定位到N223-53的235至349位之间的残基。E385也被鉴定为Exo53的一个潜在活性位点残基。
