A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-d-Glucose Dehydrogenase from Pseudomonas aureofaciens
A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-d-Glucose Dehydrogenase from Pseudomonas aureofaciens
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DOI：
10.1128/jb.02376-14
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发表时间：
2015-02
影响因子：
3.2
通讯作者：
Kiwamu Umezawa;K. Takeda;T. Ishida;Naoki Sunagawa;Akiko Makabe;K. Isobe;K. Koba;H. Ohno;M. Samejima;N. Nakamura;K. Igarashi;Makoto Yoshida
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作者：
Kiwamu Umezawa;K. Takeda;T. Ishida;Naoki Sunagawa;Akiko Makabe;K. Isobe;K. Koba;H. Ohno;M. Samejima;N. Nakamura;K. Igarashi;Makoto Yoshida
摘要 从金色假单胞菌（Pseudomonas aureofaciens）中克隆了一个编码一种类似于丝状真菌中吡咯并喹啉醌（PQQ）依赖性糖脱氢酶的基因，该基因在碳水化合物活性酶（CAZy）数据库中属于新的辅助活性（AA）家族12。所克隆酶的推导氨基酸序列与先前已鉴定的PQQ依赖性酶仅显示出较低的同源性，多序列比对分析表明，该酶缺少在已知PQQ依赖性酶中作为PQQ结合残基起作用的三个保守精氨酸残基中的一个。重组酶在大肠杆菌表达系统中异源表达以用于进一步表征。通过将脱辅基酶与PQQ和氯化钙一起孵育制备的全酶氧化形式的紫外 - 可见（UV - Vis）吸收光谱在约350 nm处显示出一个宽峰，表明该酶结合PQQ。在全酶溶液中加入2 - 酮 - D - 葡萄糖（2KG）时，在331 nm处出现一个尖锐的峰，这归因于与酶结合的PQQ的还原，而在向纯PQQ中加入2KG时未观察到任何影响。酶促测定表明，当添加PQQ和氯化钙时，在合适的电子受体（如2,6 - 二氯酚靛酚）存在下，重组酶与2KG特异性反应。反应产物的1H核磁共振（1H - NMR）分析显示2 - 酮 - D - 葡萄糖酸（2KGA）为主要产物，明确表明重组酶氧化2KG的C - 1位。因此，该酶被鉴定为一种PQQ依赖性2KG脱氢酶（Pa2KGDH）。考虑到高底物特异性，Pa2KGDH的生理功能可能是用于生产2KGA。
ABSTRACT A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. 1H nuclear magnetic resonance (1H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.