嗜热共生杆菌内消旋-2,6-二氨基庚二酸脱氢酶中Y76对辅酶偏好性影响

Effect of residue Y76 on co-enzyme specificity of meso-Diaminopimelate dehydrogenase from Symbiobacterium thermophilum

摘要:

辅酶NAD(H) 相比NADP(H) 有稳定性好、价格低廉及更广的辅酶循环方法等优势，因此在实际应用中常需将NADP(H) 赖型的脱氢酶改造成为NAD(H) 依赖型的。来源于嗜热共生杆菌Symbiobacterium thermophilum的NADP(H) 依赖型内消旋-2,6-二氨基庚二酸脱氢酶 (meso-2,6-diaminopimelate dehydrogenase, StDAPDH) 及其突变体酶是催化还原氨化合成D-氨基酸的优良催化剂，本研究试图改变其辅酶偏好性，增强其应用优势。对其晶体结构分析可知，氨基酸残基Y76距离腺嘌呤较近，R35及R36和辅酶上磷酸基团有直接相互作用。依氨基酸侧链基团性质对Y76进行了定点突变，发现不同突变子对两种辅酶的偏好性都发生了变化；对与磷酸基团直接作用的R35、R36进行的双突变R35S/R36V，导致酶对NADP+的催化活力降低；将R35S/R36V和部分Y76突变进行了组合，发现三突变组合以NAD+为辅酶时的活力均大于以NADP+为辅酶的活力，实现了辅酶偏好性转变。这些研究工作为进一步实现StDAPDH的辅酶偏好性完全转变提供依据。

关键词:氨基酸脱氢酶，内消旋-2,6-二氨基庚二酸脱氢酶，辅酶偏好性，突变

Abstract:

In industrial application of NAD(P)H-dependent dehydrogenases, NAD(H) has the advantages over NADP(H) in higher stability, lower price and wider recycling system. Recently, a meso-2,6-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) has been found to be a useful biocatalyst for the production of D-amino acids, but it requires NADP(H) as co-enzyme. To switch the co-enzyme specificity from NADP(H) to NAD(H), we studied the effect of Y76 on the co-enzyme specificity of StDAPDH, because the crystal structural analysis indicated that residue Y76 is near the adenine ring. The mutation of Y76 exerted significant effect on the co-enzyme specificity. Furthermore, the double mutant R35S/R36V significantly lowered the specific activity toward NADP+, and the combination of R35S/R36V with some of the Y76 mutants resulted in mutant enzymes favorable NAD+ over NADP+. This study should provide useful guidance for the further development of highly active NAD+-dependent StDAPDH by enzyme engineering.

Key words:amino acid dehydrogenase, meso-diaminopimelate dehydrogenase, co-enzyme preference, mutagenesis