Studies on Structure and Function of Formaldehyde Dismutase

甲醛歧化酶（FDM）是在一株恶臭假单胞菌F61中发现的，它能催化醛的歧化酶，从而生成等摩尔量的醇和酸。歧化反应中的氧化还原是由辅酶NAD(H)介导的，它牢牢地（但不是共价的）位于该酶的活性位点。这种催化氧化还原而不添加电子受体的酶是生物反应器的有利催化剂。该酶半反应的动力学参数与传统的nad1连接醇脱氢酶非常相似。FDM最显著的特性是对NAD(H)的高亲和力。为了阐明参与NAD(H)结合的蛋白结构，对FDM基因进行了克隆和表征。将FDM基因克隆到putida F61总DNA的7.2 kb Sau 3Al片段的载体质粒pKT230上。该酶的基因在pKT230 （pEC5）和ptFC19 （pEC21）中被克隆为3.2 kb的Hind III和Pst I消化片段。携带pEC5 （N5）的恶臭假单胞菌TN1126的FDM活性约为亲本菌株F61的1%。该酶蛋白是从重组菌株N5中纯化出来的，并以21%的活性产量获得了电泳均匀的蛋白。纯化酶的HPLC （TSKgel 3000SW）和SDS-PAGE蛋白与F61酶相同。纯化后的N5酶仅为1%。F61酶的结构NAD透析可恢复部分N5酶活性。作为一个初步的结论，细菌细胞必须含有高浓度的NAD或NADH才能合成活性FDM。pEC5中插入的碱基序列正在测定中。

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.