Studies on Structure and Function of Formaldehyde Dismutase

甲醛歧化酶（FDM）是在恶臭假单胞菌（Pseudomanas putida）F61中发现的一种催化醛歧化反应的酶，它能催化醛歧化反应生成等摩尔量的醇和酸。歧化反应中的氧化还原由辅酶NAD（H）介导，辅酶NAD（H）牢固地（但不是共价地）在该酶的活性位点。这种不添加电子受体而催化氧化还原的酶作为生物反应器的催化剂是有利的。该酶的半反应动力学参数与传统的NAD-连接的醇脱氢酶的动力学参数非常相似。FDM最典型的特性是对NAD（H）的高亲和力。为了阐明参与NAD（H）结合的蛋白质结构，克隆了FDM基因，将FDM基因以7.2kb的Sau 3A 1片段的形式克隆到pKT 230载体上。将负责该酶的基因作为3.2kb的Hind III和Pst I消化片段重新克隆到pKT 230（pEC 5）和ptFC 19（pEC 21）中。携带pEC 5（N5）的恶臭假单胞菌TN 1126表现出约1%的亲株F61的FDM活性。从重组菌株N5中纯化酶蛋白，并以21%的活性收率获得了一种在电泳上均一的蛋白。纯化的酶的HPLC（TSKgel 3000 SW）和SDS-PAGE蛋白与F61酶的那些一致。纯化的N5酶仅表现出1%。F61酶用NAD透析可恢复部分N5酶活性。初步结论是，细菌细胞必须含有高浓度的NAD或NADH才能合成有活性的FDM。正在确定pEC 5中插入的碱基序列。

Formaldehyde dismutase (FDM), which was found in an isolated Pseudomanas putida F61, catalyzes the dismutation of aldehydes, leading to the formation of equimolar amount of alcohols and acids. The oxidoreduction in the dismutation reaction is mediated by the coenzyme NAD(H) which firmly (but not covalently) at the active site of this enzyme. Such an enzyme that catalyzes the oxidoreduction without addition of an electron acceptor is favorable as a catalyst for a bioreactor. The kinetic parameters of the half reaction of this enzyme are very similar to those of conventional NAD-linked alcohol dehydrogenase. The most characteristic properties of FDM is the high affinity for NAD(H). In order to elucidate the protein structures participating in the binding of NAD(H), FDM gene was cloned and characterized.FDM gene was cloned onto a vector plasmid pKT230 as a 7.2 kb Sau 3Al fragment of the total DNA of P. putida F61. The gene responsible for the enzyme was recloned in pKT230 (pEC5) and ptFC19 (pEC21) as a 3.2 kb, Hind III and Pst I digest fragment. The Pseudomonas putida TN1126 harboring pEC5 (N5) exhibited the FDM activity at about 1% of the parent strain, F61. The enzyme protein was purified from the recombinant strain, N5, and obtained an electrophoretically homogeneous protein at 21% activity yield. The HPLC (TSKgel 3000SW) and SDS-PAGE proteins of the purified enzyme were identical with those of the F61 enzyme. The purified N5 enzyme exhibited only 1%. of the F61 enzyme. A part of the activity of N5 enzyme was restored by the dialysis with NAD. As to a tentative conclusion, the bacterial cells must contain a high concentration of NAD or NADH in order to synthesis of the active FDM. Determination of the base sequence of the insertion in pEC5 is in progress.