Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2
Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2
复制标题
DOI：
10.1073/pnas.1218769110
复制
发表时间：
2013-02-19
影响因子：
11.1
通讯作者：
Shanklin, John
中科院分区：
文献类型：
--
作者：
Andre, Carl;Kim, Sung Won;Shanklin, John
生物生产的烷烃是石油衍生化学品的潜在可再生替代品。由酰基载体蛋白还原酶和醛去甲酰化加氧酶(ADO)组成的蓝藻途径以氧依赖的方式通过醛中间体将酰基载体蛋白转化为相应的n-1烷烃(O-2为K-m，84+/-9muM)。在体外，ADO只翻转了三次，但在耗尽的分析中添加更多的ADO会导致额外的产物形成。在评价用于驱动ADO催化的过氧化氢分流时，我们发现过氧化氢(H_2O_2)对ADO有抑制作用，其表观K-I为16+/-6µM，且H_2O_2对O_2的抑制是混合型的。添加过氧化氢酶(CAT)恢复了ADO活性，表明抑制是可逆的，并依赖于H_2O_2，这是由于还原剂消耗与烷烃形成的弱耦合所致。动力学分析表明，长链(C14-C18)底物遵循Michaelis-Menten动力学，而中短链(C8-C12)则表现出底物抑制作用。一种双功能蛋白，包括N端的CAT和C端的ADO(CAT-ADO)，通过将其转化为共底物O-2来防止过氧化氢的抑制。事实上，当向厌氧反应混合物中加入过氧化氢时，可以观察到融合蛋白产生烷烃。在实验中，CAT-ADO的转化率是天然ADO的3倍，而CAT-ADO的转化率是225倍，并且它在大肠杆菌中的表达使每个活性部位的催化转化率比天然ADO的表达提高了5倍。我们提出了“通过抑制物代谢保护”这一术语，用于将抑制物代谢成非抑制化合物的融合蛋白。
Biologically produced alkanes represent potential renewable alternatives to petroleum-derived chemicals. A cyanobacterial pathway consisting of acyl-Acyl Carrier Protein reductase and an aldehyde-deformylating oxygenase (ADO) converts acyl-Acyl Carrier Proteins into corresponding n-1 alkanes via aldehyde intermediates in an oxygen-dependent manner (K-m for O-2, 84 +/- 9 mu M). In vitro, ADO turned over only three times, but addition of more ADO to exhausted assays resulted in additional product formation. While evaluating the peroxide shunt to drive ADO catalysis, we discovered that ADO is inhibited by hydrogen peroxide (H2O2) with an apparent K-i of 16 +/- 6 mu M and that H2O2 inhibition is of mixed-type with respect to O-2. Supplementing exhausted assays with catalase (CAT) restored ADO activity, demonstrating that inhibition was reversible and dependent on H2O2, which originated from poor coupling of reductant consumption with alkane formation. Kinetic analysis showed that long-chain (C14-C18) substrates follow Michaelis-Menten kinetics, whereas short and medium chains (C8-C12) exhibit substrate inhibition. A bifunctional protein comprising an N-terminal CAT coupled to a C-terminal ADO (CAT-ADO) prevents H2O2 inhibition by converting it to the cosubstrate O-2. Indeed, alkane production by the fusion protein is observed upon addition of H2O2 to an anaerobic reaction mix. In assays, CAT-ADO turns over 225 times versus three times for the native ADO, and its expression in Escherichia coli increases catalytic turnovers per active site by fivefold relative to the expression of native ADO. We propose the term "protection via inhibitor metabolism" for fusion proteins designed to metabolize inhibitors into noninhibitory compounds.