van Hellemond Erik W, van Dijk Marianne, Heuts Dominic P H M, Janssen Dick B, Fraaije Marco W
Laboratory of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Appl Microbiol Biotechnol. 2008 Mar;78(3):455-63. doi: 10.1007/s00253-007-1310-4. Epub 2008 Jan 9.
A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (KM=8.2 microM, kcat=26 s(-1)). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1/2 at 50 degrees C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh.
从红平红球菌NCIMB 11540的基因组中鉴定出一个编码腐胺氧化酶（PuORh，EC 1.4.3.10）的基因。该基因被克隆到pBAD载体中，并在大肠杆菌中高水平过表达。纯化后的酶显示为一种可溶性二聚体黄素蛋白，由50 kDa的亚基组成，含有非共价结合的黄素腺嘌呤二核苷酸作为辅因子。在所有底物中，发现对腐胺的催化效率最高（KM = 8.2 μM，kcat = 26 s-1）。PuORh能接受更长的多胺，而短二胺和单胺则强烈抑制其活性。PuORh是一种相当耐热的酶，在50℃下的半衰期为2小时。基于人单胺氧化酶B的晶体结构，我们构建了PuORh的模型结构，这表明Glu324对底物结合起着关键作用。该残基的突变导致催化效率急剧下降（五个数量级）。有趣的是，突变酶对野生型PuORh不接受的单胺有活性。
