Enzymology/Molecular Biology of Aldehyde Dehydrogenase

描述（由申请人提供）：有17个基因编码各种形式的人醛脱氢酶（ALDH）。尽管对一些同工酶的结构和机制了解甚多，但大多数ALDH家族成员的生理作用尚不清楚。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体，这些同工酶具有与亲本酶不同的特性。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增加活性和增强稳定性的同工酶。限速步骤是1类、2类和3类同工酶，这些酶的结构和作用机理已经被研究过，但每种酶的形式不同。我们将在任何同工酶中发现的突变引入到其他同工酶中，以确定我们是否可以增加催化过程中的相同步骤。目的是为了更多地了解控制每个同工酶的限速步骤，然后尝试使用来自不同同工酶的信息增益来提高其他同工酶的催化效率。稳定性增加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aldophophamide能力的酶，aldophophamide是一种在前药环磷酰胺转化过程中产生的有毒醛，这种化合物会破坏一些肿瘤。不幸的是，这种药物会通过氧化有毒的醛来杀死任何没有ALDH保护的细胞。骨髓就是这样一种细胞。如果我们能够产生一种具有增强抗醛磷酰胺活性的突变体，我们将使用细胞培养工作来确定用更活跃的ALDH突变体转化的细胞是否可以保护细胞免受化合物的毒性作用。

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.