硫氰酸水解酶(SCNase)是一种主要来自于硫杆菌属的含钴酶,能够催化焦化废水中的主要成分硫氰酸盐降解。它由3个亚基组成,其活性中心存在2个翻译后修饰氧化的半胱氨酸,并且和钴离子形成独特的爪型配位结构。硫氰酸水解酶在序列和机构上都与钴型腈水合酶有很高的相似性,但活性并不互通。通过在公共序列库里搜寻,发现了一种来自玫瑰单胞菌Roseomonas stagni的新基因型的硫氰酸水解酶。该研究表达、纯化并且对这个硫氰酸水解酶(Rose.s SCNase)进行了表征。有趣的是,Rose.s SCNase不但对硫氰酸钾有催化活性,对腈类底物也具有催化活性,这是迄今为止发现的第一个活性互通的天然腈类降解酶。Rose.s SCNase在最适pH和温度下对烟腈的比酶活为(17.8±1.3) U/mg,对丙烯腈的比酶活为(30.6±1.9) U/mg,对硫氰酸钾的比酶活为(13.5±0.9) U/mg。结构比对结果显示,位于Rose.s SCNase底物结合口袋顶部氨基酸的不同可能是其存在活性互通的原因。Rose.s SCNase可能对研究含氰基底物降解方面有巨大的帮助。
Thiocyanate hydrolase(SCNase) is a cobalt-containing enzyme mainly derived from Thiobacillus. They can catalyze the degradation of thiocyanate, a main component in coking wastewater. Thiocyanate hydrolase consists of three subunits. There are two post-translational oxidized cysteines in the active center, which form a unique claw-type coordination structure with cobalt ions. Although SCNase shares high homology with the Co-NHase, both in amino acid sequences and structure, they exclusively catalyze only their own substrates. We discovered a new genotype of thiocyanate hydrolase by searching in the public sequence database. In this study, we expressed, purified, and characterized this thiocyanate hydrolase (Rose.s SCNase). Interestingly, Rose.s SCNase had catalytic activity not only against potassium thiocyanate, but also against nitrile substrates. It is the first natural nitrile degrading enzyme discovered so far. The results of structural comparison showed that the different amino acids at the top of the substrate binding pocket of Rose.s SCNase might be the reason of the interactivity. Rose.s SCNase will be a great help to our research on the degradation of cyano-containing substrates.
[1] KOBAYASHI M, SHIMIZU S.Metalloenzyme nitrile hydratase:Structure, regulation, and application to biotechnology[J].Nature Biotechnology, 1998, 16(8):733-736.
[2] ASANO Y, TANI Y, YAMADA H.A new enzyme “nitrile hydratase” which degrades acetonitrile in combination with amidase[J].Agricultural and Biological Chemistry, 1980, 44(9):2251-2252.
[3] CHEN J, ZHENG R C, ZHENG Y G, et al.Microbial transformation of nitriles to high-value acids or amides[J].Advances in Biochemical Engineering/Biotechnology, 2009, 113:33-77.
[4] NAGASAWA T, MATHEW C D, MAUGER J, et al.Nitrile hydratase-catalyzed production of nicotinamide from 3-cyanopyridine in Rhodococcus rhodochrous J1[J].Applied and Environmental Microbiology, 1988, 54(7):1766-1769.
[5] PRASAD S, BHALLA T C.Nitrile hydratases (NHases):At the interface of academia and industry[J].Biotechnology Advances, 2010, 28(6):725-741.
[6] NELP M T, SONG Y, WYSOCKI V H, et al.A protein-derived oxygen is the source of the amide oxygen of nitrile hydratases[J].Journal of Biological Chemistry, 2016, 291(15):7822-7829.
[7] KOVACS J A.Synthetic analogues of cysteinate-ligated non-heme iron and non-corrinoid cobalt enzymes[J].Chemical Reviews, 2004, 104(2):825-848.
[8] YAMADA H, KOBAYASHI M.Nitrile hydratase and its application to industrial production of acrylamide[J].Bioscience, Biotechnology, and Biochemistry, 1996, 60(9):1391-1400.
[9] HOURAI S, MIKI M, TAKASHIMA Y, et al.Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii[J].Biochemical and Biophysical Research Communications, 2003, 312(2):340-345.
[10] WU S, FALLON R D, PAYNE M S.Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli:Activity requires a novel downstream protein[J].Applied Microbiology and Biotechnology, 1997, 48(6):704-708.
[11] NOJIRI M, YOHDA M, ODAKA M, et al.Functional expression of nitrile hydratase in Escherichia coli:Requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine[J].J The Journal of Biochemistry, 1999, 125(4):696-704.
[12] HASHIMOTO Y, NISHIYAMA M, HORINOUCHI S, et al.Nitrile hydratase gene from Rhodococcus sp.N-774 requirement for its downstream region for efficient expression[J].Bioscience, Biotechnology, and Biochemistry, 1994, 58(10):1859-1865.
[13] MARTINEZ S, YANG X H, BENNETT B, et al.A cobalt-containing eukaryotic nitrile hydratase[J].Biochimica et Biophysica Acta.Proteins and Proteomics, 2017, 1 865(1):107-112.
[14] MURAKAMI T, NOJIRI M, NAKAYAMA H, et al.Post-translational modification is essential for catalytic activity of nitrile hydratase[J].Protein Science:A Publication of the Protein Society, 2000, 9(5):1024-1030.
[15] CHENG Z Y, XIA Y Y, ZHOU Z M.Recent advances and promises in nitrile hydratase:From mechanism to industrial applications[J].Frontiers in Bioengineering and Biotechnology, 2020, 8:352.
[16] TSUJIMURA M, DOHMAE N, ODAKA M, et al.Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp.N-771.Evidence of a novel post-translational modification in the cysteine ligand[J].The Journal of Biological Chemistry, 1997, 272(47):29454-29459.
[17] NOGUCHI T, NOJIRI M, TAKEI K I, et al.Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy[J].Biochemistry, 2003, 42(40):11642-11650.
[18] ARAKAWA T, KAWANO Y, KATAOKA S, et al.Structure of thiocyanate hydrolase:A new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center[J].Journal of Molecular Biology, 2007, 366(5):1497-1509.
[19] KATAYAMA Y, HASHIMOTO K, NAKAYAMA H, et al.Thiocyanate hydrolase is a cobalt-containing metalloenzyme with a cysteine-sulfinic acid ligand[J].Journal of the American Chemical Society, 2006, 128(3):728-729.
[20] YAMANAKA Y, ARAKAWA T, WATANABE T, et al.Two arginine residues in the substrate pocket predominantly control the substrate selectivity of thiocyanate hydrolase[J].Journal of Bioscience and Bioengineering, 2013, 116(1):22-27.
[21] XIA Y Y, PEPLOWSKI L, CHENG Z Y, et al.Metallochaperone function of the self-subunit swapping chaperone involved in the maturation of subunit-fused cobalt-type nitrile hydratase[J].Biotechnology and Bioengineering, 2019, 116(3):481-489.
[22] ZHOU Z M, HASHIMOTO Y, CUI T W, et al.Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase:Intermediates and a proposed mechanism for self-subunit swapping maturation[J].Biochemistry, 2010, 49(44):9638-9648.
[23] GUO J L, CHENG Z Y, BERDYCHOWSKA J, et al.Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase[J].International Journal of Biological Macromolecules, 2021, 181:444-451.
[24] CHENG Z Y, JIANG S J, ZHOU Z M.Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles[J].Biochemical and Biophysical Research Communications, 2021, 575:8-13.
[25] 张晓欢, 崔文璟, 周哲敏.高分子量腈水合酶在大肠杆菌中的表达策略及重组菌的细胞催化[J].微生物学通报, 2016, 43(10):2121-2128.
ZHANG X H, CUI W J, ZHOU Z M.Strategy of high molecular mass nitrile hydratase expression in Escherichia coli and the whole-cell catalysis by the recombinant strains[J].Microbiology China, 2016, 43(10):2121-2128.
[26] 张赛兰, 李婷, 程中一, 等.新型耐热腈水合酶的异源表达及其催化工艺研究[J].食品与发酵工业, 2020, 46(14):108-113.
ZHANG S L, LI T, CHENG Z Y, et al.Heterologous expression of a novel thermostable nitrile hydratase and its catalytic process[J].Food and Fermentation Industries, 2020, 46(14):108-113.
