Department of Chemistry, Barnard College, 3009 Broadway, New York, NY 10027, USA.
Department of Biology, Barnard College, 3009 Broadway, New York, NY 10027, USA; Data Science Institute Columbia University, New York, NY 10027 USA.
J Inorg Biochem. 2021 Jun;219:111409. doi: 10.1016/j.jinorgbio.2021.111409. Epub 2021 Mar 16.
Interest in understanding the environmental distribution of the alkane monooxygenase (AlkB) enzyme led to the identification of over 100 distinct alkane monooxygenase (AlkB) enzymes containing a covalently bound, or fused, rubredoxin. The rubredoxin-fused AlkB from Dietzia cinnamea was cloned as a full-length protein and as a truncated protein with the rubredoxin domain deleted. A point mutation (V91W) was introduced into the full-length protein, with the goal of assessing how steric bulk in the putative substrate channel might affect selectivity. Based on activity studies with alkane and alkene substrates, the rubredoxin-fused AlkB oxidizes a similar range of alkane substrates relative to its rubredoxin domain-deletion counterpart. Oxidation of terminal alkenes generated both an epoxide and a terminal aldehyde. The products of V91W-mutant-catalyzed oxidation of alkenes had a higher aldehyde-to-epoxide ratio than the products formed in the presence of the wild type protein. These results are consistent with this mutation causing a structural change impacting substrate positioning.
人们对了解烷烃单加氧酶 (AlkB) 酶的环境分布很感兴趣，这导致了 100 多种不同的烷烃单加氧酶 (AlkB) 酶被鉴定出来，这些酶含有一个共价结合的或融合的 rubredoxin。来自食红红球菌的 rubredoxin 融合 AlkB 被克隆为全长蛋白和缺失 rubredoxin 结构域的截断蛋白。在全长蛋白中引入了一个点突变 (V91W)，目的是评估假定的底物通道中的空间位阻如何影响选择性。基于烷烃和烯烃底物的活性研究，rubredoxin 融合的 AlkB 相对于其 rubredoxin 结构域缺失的对应物氧化了类似范围的烷烃底物。末端烯烃的氧化生成了环氧化物和末端醛。V91W 突变体催化烯烃氧化的产物的醛与环氧化物的比值高于在野生型蛋白存在下形成的产物。这些结果与该突变导致影响底物定位的结构变化一致。
