Enzymology/Molecular Biology of Aldehyde Dehydrogenase

描述(申请人提供)：有17个基因编码各种形式的人醛脱氢酶(ALDH)。ALDH家族中大多数成员的生理作用尚不清楚，尽管人们对一些同工酶的结构和机制知道很多。一种容易出错的PCR突变方法使我们能够产生一些同工酶的突变，这些突变的性质与亲本酶的性质不同。我们将在这一发现的基础上，使用突变方法来选择与亲本酶相比具有更高活性和更高稳定性的同工酶。限速步骤是1类、2类和3类同工酶，这些酶的结构和机制已经被研究过，每种酶的形式都不同。我们将把发现的任何一种同工酶的突变引入其他同工酶中，以确定我们是否可以在催化过程中增加相同的步骤。其目的既是为了更多地了解是什么控制了每个同工酶的限速步骤，然后试图利用从不同同工酶获得的信息来提高其他同工酶的催化效率。稳定性提高的同工酶将从筛选过程中鉴定出来，然后在体外和体内进行研究，目的是产生更稳定的酶。此外，我们将尝试选择一种具有增强氧化能力的酶，该酶是一种在前药物环磷酰胺转化过程中产生的有毒醛，环磷酰胺是一种可以摧毁一些肿瘤的化合物。不幸的是，这种药物会杀死任何没有ALDH的细胞，通过氧化有毒的醛来保护它。骨髓就是这样一种细胞。如果我们能培育出一种抗醛磷酰胺活性增强的突变体，我们将利用细胞培养工作来确定转化了更活跃的突变体ALDH的细胞是否能保护细胞免受化合物的毒性影响。

DESCRIPTION (provided by applicant): There are 17 genes coding for various forms of human aldehyde dehydrogenase (ALDH). The physiological role of most members of the ALDH family is not known though much is known about the structure and mechanisms of a few of the isozymes. An error prone PCR mutational approach has allowed us to produce mutants of some of the isozymes that have properties that differ from those of the parent enzyme. We will build upon this finding to use the mutational approach to select for isozymes that have increased activity as well as enhanced stability as compared to the parent enzyme. The rate-limiting step is the class 1, 2 and 3 isozymes, enzymes whose structure and mechanism have been studied, are different in each enzyme form. We will introduce the mutations found with any one isozyme into the others to determine if we can increase the same step in the catalytic process. The purpose is both to learn more about what controls the rate limiting step in each isozyme and then try to improve the catalytic efficiencies in the other isozymes using information gain from a different isozyme. Isozymes with increased stability will be identified from the screening procedure and then studied both in vitro and in vivo with the goal of producing a more stable enzyme. In addition we will try to select for an enzyme with an enhanced ability to oxidize aldophophamide, a toxic aldehyde produced during the transformation of the pro-drug cyclophosphamide, a compound that will destroy some tumors. Unfortunately, the drug will kill any cell that does not have an ALDH to protect it by oxidizing the toxic aldehyde. Bone marrow is one such cell. If we can produce a mutant with enhanced activity against aldophophamide we will use cell culture work to determine if cells transformed with the more active mutant ALDH can protect the cell from the toxic effects of the compound.