STRUCTURE, FUNCTION, & GENETICS OF E COLI PHOSPHOTRIESTERASE HOMOLOG

抗体可以被制成具有酶的功能，因为它们 拥有稳定的结构框架，可以支持许多 不同的结合位点突变。 这允许对 各种化学环境，包括那些 易受化学催化的影响。 大自然选择了 α/β-桶折叠作为一个类似的适应性框架， 新酶的分化进化。 大约10%的已知 酶是α/β桶家族的成员， 酶活性、催化效率和辅因子的使用变化 广泛传播 磷酸三酯酶（PTE）是一种来源于土壤的a/B桶酶 催化水解和解毒的细菌 磷酸三酯杀虫剂和神经毒剂。 酶活性位点 含有两个锌原子与His侧链结合到蛋白质上， 由氨甲酰化的Lys侧链桥接。 这种酶非常 高效;磷酸三酯酶的催化速率仅受以下因素的限制： 酶和底物在溶液中的扩散速率。 因为 这种酶的磷酸三酯底物是合成化合物 由于没有已知的生物学对应物， 导致PTE发明的进化途径-- 这个新的活动是如何开始的？ 使用 同源性搜索技术，我们发现了一个E.大肠杆菌基因组开放 与磷酸三酯酶高度相似的阅读框（ORF）。 该ORF位于E.大肠杆菌染色体 一组新发现的ORF似乎编码了 在有机磷代谢中。 我们将其命名为E。大肠杆菌同源物 磷酸三酯酶同源蛋白（PHP）。 的多肽序列 PHP与PTE有28%的相同性， PTE中Zn原子的配体在PHP中是保守的。 此外，PHP 在对应的氨基酸残基的位置上含有Glu残基。 PTE的氨甲酰化赖氨酸。 基于这种结构同源性，我们 预测：1）PHP具有a/B桶结构; 2）PHP是 3）PHP是一种金属酶; 4）PHP是 PTE起源的酶亚家族。 的关系 PTE和PHP提供了一个独特的研究机会， 酶的进化。 PHP代表起点，PTE代表终点 问题是，我们是否可以重建 在实验室中将PHP转化为PTE的突变。 我们也 对了解PHP的自然生物功能感兴趣， 这可能提供线索，为什么PHP亚家族成员被招募， 土壤细菌成为一种新的磷酸三酯水解酶。

Antibodies can be made to function as enzymes bec ause they possess a stable structural framework that can support a number of different combining site mutations. This allows for sampling of a wide variety of chemical environments, including those that are amenable to chemical catalysis. Nature has selected the alpha/beta-barrel fold as a similar adaptable framework for the divergent evolution of new enzymes. Approximately 10% of all known enzymes are members of the alpha/beta barrel family and their enzymatic activities, catalytic efficiencies, and cofactor usage vary widely. Phosphotriesterase (PTE) is an a/b barrel enzyme from soil bacteria which catalyzes the hydrolysis and detoxification of phosphotriester insecticides and nerve agents. The enzyme active site contains two zinc atoms bound to the protein with His side chains and bridged by a carbamoylated Lys sidechain. The enzyme is remarkably efficient; the catalytic rate of phosphotriesterase is limited only by the diffusion rate of enzyme and substrate in solution. Because the phosphotriester substrates for this enzyme are synthetic compounds with no known biological counterpart, it is interesting to consider the evolutionary pathway that led to the invention of PTE--what was the starting point and how was this new activity created? Using homology searching techniques, we have found an E. coli genomic open reading frame (ORF) that is highly similar to phosphotriesterase. This ORF is located at about 74 min. on the E. coli chromosome in a cluster of newly identified ORF's that appear to encode enzymes used in organophosphate metabolism. We have named the E. coli homolog phosphotriesterase homology protein (PHP). The polypeptide sequence of PHP is 28% identical to PTE and the four His residues that are ligands to the Zn atoms in PTE are conserved in PHP. In addition, PHP contains a Glu residue at the corresponding position of the carbamoylated Lys of PTE. Based on this structural homoloy, we predict that: 1) PHP has an a/b barrel structure; 2) PHP is an enzyme; 3) PHP is a metallo enzyme; and 4) PHP is a member of the enzyme subfamily from which PTE originated. The relationship between PTE and PHP presents a unique research opportunity to study a pathway in enzyme evolution. PHP represents the start point and PTE the end point, and the question is whether we can recreate the mutations in the laboratory that convert PHP into PTE. We are also interested in understanding the natural biological function of PHP, as this may provide clues to why a PHP subfamily member was recruited by soil bacteria to become a new phosphotriester hydrolase.