Heckmann Christian M, Gourlay Louise J, Dominguez Beatriz, Paradisi Francesca
School of Chemistry, University of Nottingham, Nottingham, United Kingdom.
Department of Biosciences, Università degli Studi di Milano, Milan, Italy.
Front Bioeng Biotechnol. 2020 Jul 22;8:707. doi: 10.3389/fbioe.2020.00707. eCollection 2020.
The identification and 3D structural characterization of a homolog of the ()-selective transaminase (RTA) from (RTA), from the thermotolerant fungus (RTA) is here reported. The thermostability of RTA (40% retained activity after 7 days at 40°C) was initially attributed to its tetrameric form in solution, however subsequent studies of RTA revealed it also exists predominantly as a tetramer yet, at 40°C, it is inactivated within 48 h. The engineering of a cysteine residue to promote disulfide bond formation across the dimer-dimer interface stabilized both enzymes, with RTA_G205C retaining almost full activity after incubation at 50°C for 7 days. Thus, the role of this mutation was elucidated and the importance of stabilizing the tetramer for overall stability of RTAs is highlighted. RTA accepts the common amine donors ()-methylbenzylamine, isopropylamine, and d-alanine as well as aromatic and aliphatic ketones and aldehydes.
本文报道了从耐热真菌中鉴定出的()-选择性转氨酶(RTA)的同源物，并对其进行了三维结构表征。RTA的热稳定性(在40°C下7天后保留40%的活性)最初归因于其在溶液中的四聚体形式，然而，随后对RTA的研究表明，它也主要以四聚体形式存在，但在40°C下，它会在48小时内失活。通过工程改造半胱氨酸残基以促进二聚体-二聚体界面形成二硫键，使两种酶都得到了稳定，RTA_G205C在50°C下孵育7天后仍保留几乎全部活性。因此，阐明了这种突变的作用，并强调了稳定四聚体对RTA整体稳定性的重要性。RTA接受常见的胺供体()-甲基苄胺、异丙胺和d-丙氨酸以及芳香族和脂肪族酮和醛。
