Pestov Nikolay B, Kalinovsky Daniel V, Larionova Tatyana D, Zakirova Alia Z, Modyanov Nikolai N, Okkelman Irina A, Korneenko Tatyana V
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
Department of Physiology and Pharmacology, University of Toledo, Toledo, OH, United States of America.
PeerJ. 2019 Apr 5;7:e6691. doi: 10.7717/peerj.6691. eCollection 2019.
Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.
LOX open reading frame was cloned from in an expression vector. Recombinant lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting.
Cultured has no detectable amine oxidase activity. HTU-LOX may be expressed in with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in cells.
contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.
This is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.
赖氨酰氧化酶（LOX）在哺乳动物中已得到广泛研究，而原核基因组中最近发现的同源物的特性和功能仍不清楚。
从 中克隆LOX开放阅读框并插入表达载体。重组赖氨酰氧化酶（HTU-LOX）蛋白在变性条件下通过金属亲和层析纯化，然后复性。在辣根过氧化物酶存在下，通过荧光法测定胺氧化酶活性，以过氧化氢释放量与10-乙酰-3,7-二羟基吩恶嗪氧化量的耦合来衡量。制备兔多克隆抗体并用于蛋白质印迹分析。
培养的 没有可检测到的胺氧化酶活性。HTU-LOX可能在 中以高蛋白产量表达。全长蛋白没有催化活性。因此，我们推测疏水的N端区域可能会干扰正确折叠，去除该区域可能有益。实际上，在变性条件下纯化的缺失N端疏水信号肽的截短的His标签HTU-LOX可以成功复性为活性酶，更大的N端截短进一步提高了胺氧化酶活性。在pH 6.2的铜存在下复性最佳，且对盐不敏感。HTU-LOX对LOX抑制剂3-氨基丙腈敏感。HTU-LOX可使哺乳动物LOX的常见底物如含赖氨酸的多肽和聚合物脱氨。HTU-LOX与哺乳动物LOX的主要区别在于前者底物特异性较宽松。HTU-LOX容易氧化各种伯胺，包括牛磺酸、甘氨酸等化合物，苄胺是较差的底物。值得注意的是，HTU-LOX对几种氨基糖苷类抗生素和多粘菌素也有活性。蛋白质印迹分析表明，抗HTU-LOX多克隆抗体的表位与 细胞中的一种高分子量蛋白一致。
含有一个赖氨酰氧化酶基因，该基因经异源表达产生一种活性重组酶，具有所有已知LOX共有的重要生化特性，例如对3-氨基丙腈敏感。然而，其在宿主中的天然功能似乎是隐藏的。
这是关于古菌赖氨酰氧化酶某些特性的首次报道，也是远距离分类群之间假设的水平转移后酶特性进化的一个有趣例子。
