van Oosterwijk Niels, Willies Simon, Hekelaar Johan, Terwisscha van Scheltinga Anke C, Turner Nicholas J, Dijkstra Bauke W
Laboratory of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
School of Chemistry, Manchester Institute of Biotechnology, University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
Biochemistry. 2016 Aug 9;55(31):4422-31. doi: 10.1021/acs.biochem.6b00370. Epub 2016 Jul 29.
ω-Transaminases are enzymes that can introduce an amino group in industrially interesting compounds. We determined crystal structures of two (S)-selective ω-transaminases, one from Arthrobacter sp. (Ars-ωTA) and one from Bacillus megaterium (BM-ωTA), which have 95% identical sequences but somewhat different activity profiles. Substrate profiling measurements using a range of (R)- and (S)-substrates showed that both enzymes have a preference for substrates with large, flat cyclic side groups, for which the activity of BM-ωTA is generally somewhat higher. BM-ωTA has a preference for (S)-3,3-dimethyl-2-butylamine significantly stronger than that of Ars-ωTA, as well as a weaker enantiopreference for 1-cyclopropylethylamine. The crystal structures showed that, as expected for (S)-selective transaminases, both enzymes have the typical transaminase type I fold and have spacious active sites to accommodate largish substrates. A structure of BM-ωTA with bound (R)-α-methylbenzylamine explains the enzymes' preference for (S)-substrates. Site-directed mutagenesis experiments revealed that the presence of a tyrosine, instead of a cysteine, at position 60 increases the relative activities on several small substrates. A structure of Ars-ωTA with bound l-Ala revealed that the Arg442 side chain has been repositioned to bind the l-Ala carboxylate. Compared to the arginine switch residue in other transaminases, Arg442 is shifted by six residues in the amino acid sequence, which appears to be a consequence of extra loops near the active site that narrow the entrance to the active site.
ω-转氨酶是一类能够在具有工业应用价值的化合物中引入氨基的酶。我们测定了两种（S）-选择性ω-转氨酶的晶体结构，一种来自节杆菌属（Ars-ωTA），另一种来自巨大芽孢杆菌（BM-ωTA），它们的序列有95%相同，但活性谱略有不同。使用一系列（R）-和（S）-底物进行的底物谱分析测量表明，这两种酶都偏好具有大的平面环状侧基的底物，其中BM-ωTA的活性通常略高。BM-ωTA对（S）-3,3-二甲基-2-丁胺的偏好明显强于Ars-ωTA，对1-环丙基乙胺的对映体选择性则较弱。晶体结构表明，正如（S）-选择性转氨酶所预期的那样，这两种酶都具有典型的I型转氨酶折叠结构，并且有宽敞的活性位点以容纳较大的底物。BM-ωTA与结合的（R）-α-甲基苄胺的结构解释了该酶对（S）-底物的偏好。定点诱变实验表明，在第60位存在酪氨酸而非半胱氨酸会增加对几种小底物的相对活性。Ars-ωTA与结合的l-丙氨酸的结构表明Arg442侧链已重新定位以结合l-丙氨酸的羧酸盐。与其他转氨酶中的精氨酸开关残基相比，Arg442在氨基酸序列中偏移了六个残基，这似乎是活性位点附近额外环结构使活性位点入口变窄的结果。
