3-D forms link antibiotic resistance and brain disease
3D构型相关的抗生素耐药与大脑疾病
St. Jude uses computer-generated images of different types of same enzyme to unlock mysteries of antibiotic resistance and a rare form of brain degeneration
St. Jude儿童医院的研究者利用电脑生成同一种酶的不同类型图像，并利用这些图像来解开抗生素耐药和一种罕见的大脑退化症之谜。
The story of what makes certain types of bacteria resistant to a specific antibiotic has a sub-plot that gives insight into the cause of a rare form of brain degeneration among children, according to investigators at St. Jude Children's Research Hospital. The story takes a twist as key differences among the structures of its main molecular characters disappear and reappear as they are assembled in the cell.
根据St. Jude儿童研究医院的科学家调查所得，关于某些类型细菌对一种特定抗生素具有抗性这一故事中存在一个次要情节，即它让我们深入的了解了一种罕见的儿童大脑退化症的致病原因。当这些分子在细胞内装配时，随着这些结构间的主要分子特征的关键性差异消失，重现，这个故事发生了转折。
A report on this study appears in the August 16 issue of the journal Structure.
关于这项研究的报告发表在今年8月份第16期《结构》杂志（the journal Structure.）上。
The story is based on a study of the 3-D structure of an enzyme called pantothenate kinase, which triggers the first step in the production coenzyme A (CoA), a molecule that is indispensable to all forms of life. Enzymes are proteins that speed up biochemical reactions. CoA plays a pivotal role in the cells' ability to extract energy from fatty acids and carbohydrates; bacteria need CoA to make their cell walls. The job of pantothenate kinase is to grab a molecule of pantothenic acid (vitamin B-5) and another molecule that contains a chemical group called "phosphate." The enzyme then removes the phosphate group from that molecule and sticks it onto pantothenic acid.
整个故事基于泛酸激酶的三维结构的研究，它触发辅酶A产生过程的第一步，辅酶A对于所有生命来说都是不可缺少的。酶是一种有催化功能的蛋白质，可以加速生物化学反应。辅酶A对于细胞从脂肪和碳水化合物中获取能量这一过程中扮演了重要角色，细菌需要辅酶A来构建细胞壁。泛酸激酶的工作就是捕获名为泛酸的分子（维生素B5）和另一种含有磷酸化学基团的分子。然后泛酸激酶将后者的磷酸基团移除并把磷酸基团“粘在”泛酸上。
In humans, certain mutations in this enzyme block its ability to put the phosphate group onto pantothenic acid. That diminishes the production of CoA by this route and causes the neurodegenerative disease called pantothenate kinase associated neurodegeneration (PKAN), according to Suzanne Jackowski, Ph.D., a member of the St. Jude Department of Infectious Diseases and a co-author of the paper. "We also know that certain antibiotics called pantothenamides work by impersonating vitamin B-5 and slipping into the enzyme," Jackowski said. "This blocks the bacteria's ability to produce fatty acids." The researchers already knew that different types of bacteria build their own versions of the enzyme pantothenate kinase, which are called Types I, II and III. For example, bacteria called Escherichia coli, found in the intestines and polluted water, produce Type I; Staphylococcus aureus, which causes skin infections and serious blood infections, makes type II; and Pseudomonas aeruginosa, which is an important cause of hospital-based infections, especially in burn patients, makes Type III. Types I, II and III each consist of two identical molecules called monomers, which bind together to form the enzyme. The groups had previously identified the structure and role of the Type I enzymes in pantothenamide inhibition of bacterial growth. What intrigued the St. Jude investigators now was the mystery of how Types II and III manage to do the same job even though they are constructed so differently; and why bacteria with the Type III enzyme are resistant to pantothenamide antibiotics. They also wanted to better understand the cause of PKAN in humans by comparing bacterial pantothenate kinase with the various types found in humans.
Suzanne Jackowski，理学博士，St. Jude儿童医院感染科成员，该项研究的共同作者。根据他的研究显示在人体内，该酶的某一突变将阻碍它把磷酸基团“粘在”泛酸上。这将减少辅酶A的产生，进而导致名为泛酸激酶相关的神经退化症（PKAN）、“我们还知道一类名为pantothenamides的抗生素可模仿维生素B5滑入这个酶中，” Jackowski说道，“这将妨害细菌产生脂肪酸的能力。”研究者已知不同类型的细菌有自己特有的泛酸激酶，称为类型I，类型II，类型III。例如，常见于肠道和污水中的大肠杆菌产生类型I的泛酸激酶，导致皮肤感染和严重血液感染的金黄色葡萄球菌产生类型II，重要的医源感染菌，尤见于烧伤病人的铜绿假单胞菌产生类型III。类型I,II,III都包括两个相同的单体，它们彼此结合形成了这个酶。这个研究小组此前已经鉴定了类型I的泛酸激酶的结构及其在pantothenamide抑制细菌生长过程中作用。St. Jude医院的研究者目前的计划是揭示如下几个未解之迷：类型II和类型III即使结构如此不同，它们是如何行使同样的功能的；为什么含有类型III的细菌对pantothenamide这种抗生素具有耐药性。他们还想通过比较细菌中的泛酸激酶和在人体中发现的泛酸激酶来更好的理解人泛酸激酶相关的神经退化症。
"Like all proteins, these enzymes are made up of long chains of amino acids, like beads on a string, and each type of amino acid has a unique shape and size," said Stephen White, D.Phil., chair of the St. Jude Department of Structural Biology and a co-author of the paper. The pantothenate kinase enzymes consist of two strands of amino acids that fold into various twists and turns to make a complex 3-D structure, White said. These modules, called monomers, snap together to form the enzyme. The researchers used a technique called X-ray crystallography to produce 3-D images of Types II and III and their interactions with panthothenic acid and ATP, a molecule that supplies the phosphate that the enzyme puts onto pantothenic acid.
Stephen White，理学博士., St. Jude结构生物学系主任和这篇文章的共同作者，他们说：“和所有蛋白一样，这些酶都由一长串氨基酸序列组成，如同项链上的玻璃珠一样。每一类型氨基酸都由自己独特的外形和大小，泛酸激酶包含两条氨基酸链，经过折叠扭曲形成复杂的三维结构，这些分子称为单体，彼此咬合形成这个酶。研究者利用x衍射晶体学技术产生了类型II和类型III的三维图像以及它们与泛酸和ATP（提供磷酸基团的分子）相互作用时的三维图像。
First, the researchers crystallized a sample of the enzyme and bombarded it with X-rays using the facilities at the Argonne National Laboratory in Illinois. Then they used the pattern formed by the beams as they bounced off the crystals to create computer-generated, 3-D images of the patterns of twisting and folding amino acid chains that make up the different types of pantothenate kinase and their interactions with the other molecules.
首先研究者结晶了一些酶的样本，使用位于伊利诺斯州Argonne国家实验室的设备利用X射线轰击。然后从晶体的反射光形成的图像出发，通过电脑生成组成不同类型泛酸激酶的折叠扭曲的氨基酸分子链的三维图像以及它们与其他分子相互作用时形成的三维图像。
"These images added a fascinating twist to the story of the enzymes," White said. When they studied the images, the St. Jude team realized that the monomers making up each type of enzyme were made from quite different 'strings' of amino acids. But they fold up into virtually identical looking 3-D monomers. "It was as if the uniqueness of each structure disappeared--each string folded up into the same shape as the other ones," White said. "This is very surprising because the different amino acids on each string have different sizes and different biochemical characteristics. So it would usually be impossible for them to form the same three-dimensional shapes."
White说，这些图像为这个酶的故事呈现了令人迷醉的转折。”研究小组发现组成每种酶的单体由相当不同的氨基酸链组成。但是折叠成实际上相同外形的三维单体。”White还说：“好像结构的独特性消失了似的，每一条链都折叠成与其他链相同的外形，这非常令人吃惊，因为不同氨基酸链上的氨基酸有着不同的大小的生物化学性质，因此形成相同的三维外形通常是不可能的。”
But the twist to the story did not stop there. The identically shaped monomers in each pair bind to each other in novel ways to make two versions of the same enzyme that do not look alike and yet perform the same job differently. "In other words, the differences in the 'beads on a string' shapes that disappear when the strings fold into monomers suddenly reappear when the monomers combine to form even larger structures," White said. He explained that the genes for both types of enzyme evolved from a common gene ancestor. That common gene evolved so that the final Type II and III enzyme structures look and work differently, but can still do the same job--no matter what their amino acid chains look like.
这个故事的转折还没有结束。具有相同外形的单体对彼此以新颖的方式结合使得同一种酶又两种版本，它们看起来不一样，但却行使相同的功能。“换句话说，‘项链’上不同之处消失了的‘玻璃珠’当‘项链’折叠成单体后，不同之处又重现了。”他解释道，编码不同类型酶的基因进化自同一个祖先基因。祖先基因的金华使得最终的类型II和类型III酶的外形和工作方式如此不同，但它们却从事同样的工作，无论它们的氨基酸链是怎样的。
"These images explained how the different types of the enzyme did the same job in different ways," said Mi Kyung Yun, M.S., research scientist in the St. Jude Structural Biology department and co-first author of the paper. "For example, the images showed that pantothenic acid binds to the Type III enzyme first, followed by ATP," said Yun, who was one of the investigators who created the X-ray crystallography images of the enzymes. "But with Type II enzyme, the ATP enters the enzyme from one direction, while pantothenic acid enters from another direction, in no particular sequence."
Mi Kyung Yun, St. Jude结构生物学系的科学家，这篇文章的共同第一作者，X衍射图像生成研究者之一，他说：“这些图像解释了不同类型的酶是怎样以不同的方式行使同样的功能的，例如，这些图像展示了泛酸首先与类型III酶结合，后面尾随这ATP。但是对于类型II酶，ATP从一个方向进入酶中，与此同时泛酸则从另一个方向进入酶中，并无特定的顺序。
The images also suggested that Type II enzyme in Staphylococcus aureus has a "hole" within the loops and twists of its amino acid chains that allow pantothenamide antibiotics to slip inside the enzyme, White noted. But the Type III enzyme of Pseudomonas does not have this hole, so the antibiotic cannot slip into the enzyme. A further study confirmed that the structure of Type III made Pseudomonas resistant to the antibiotics, according to Roberta Leonardi, Ph.D., a postdoctoral fellow in Jackowski's laboratory and the paper's senior author. Leonardi removed the gene for the Type I enzyme from Escherichia coli, which is normally sensitive to the antibiotic and replaced it with the gene for the Type III enzyme used by Pseudomonas. "The gene for the Type III enzyme made Escherichia coli resistant to the antibiotics," Leonardi said. "This showed that our 3-D images of the enzymes correctly predicted that pantothenamide antibiotics couldn't get into the Type III enzyme."
White提到，这些图像还暗示了金黄色葡萄球菌中的类型II酶存在一个“洞”，内有环状和扭曲的氨基酸链。这一结构允许pantothenamide抗生素滑入酶中。根据他的研究假单胞菌中的类型III酶不存在这个“洞”，因此pantothenamide抗生素久不能滑入这个酶。Roberta Leonardi,理学博士，Jackowski实验室的博士后，这篇文章的第一作者。根据他的一项深入研究证实了类型III酶使得假单胞菌对Pseudomonas抗生素具有耐药性，他敲除了对这种抗生素比较敏感的大肠杆菌中编码类型I酶，并用假单胞菌中编码类型III的基因取而代之。“编码类型III酶的基因使得大肠杆菌对这种抗生素具有了耐药性，这表明我们关于这个酶的三维图像正确地预测了Pseudomonas抗生素不能进入类型III酶”
In addition, test tube studies of the enzyme showed that Types I and II enzymes required different minerals than Type III in order to work. "One of the discoveries was that the Type III enzyme absolutely required potassium chloride, whereas Types I and II did not," Leonardi said. The study also showed that Type II pantothenate kinase in bacteria is similar to the human version, PanK2, according to Jackowski. Therefore, the structure of the Type II enzyme helps to explain how specific mutations in PanK2 disable this enzyme and cause the neurodegeneration disease called PKAN. "This holds promise that such insights will one day lead to the development of drugs designed to prevent or treat this disease," Jackowski said.
另外在试管中进行该酶的研究表明，为了正常行使功能，与III型酶相比，I型和II型酶需要不同的矿物元素。“其中一个发现是类型III酶必需氯化钾，而类型I和II则不是必需。” Leonardi说道。根据Jackowski研究还显示细菌中类型II泛酸激酶与人类中的该酶的版本（PanK2）很相似。类型II酶的结构有助于解释PanK2上发生怎样的突变会使这种酶的功能丧失以及导致名为PKAN的神经退化症。“这样的发现给了我们希望，有朝一日，我们可以设计出一种药物来阻止或治疗这种疾病，”Jackowski说道。
