Knapp Madison, Jo Minshik, Henthorn Courtney L, Brimberry Marley, Gnann Andrew D, Dowling Daniel P, Bridwell-Rabb Jennifer
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Chemistry, University of Massachusetts Boston, Boston, Massachusetts 02125, United States.
ACS Bio Med Chem Au. 2024 Nov 20;4(6):353-370. doi: 10.1021/acsbiomedchemau.4c00089. eCollection 2024 Dec 18.
Chlorophyll (Chl) is one of Nature's most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the chlorophyllase isoform CLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that CLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.
叶绿素（Chl）是自然界中生物合成和衍生化最复杂的色素之一。这种色素对生物生存至关重要，但如果过量产生或从保护性蛋白质支架中释放出来，又会具有毒性。因此，在大量生产叶绿素的同时，生物体也会投入机制来控制其降解和循环利用。参与这些后续过程的一种重要酶是叶绿素酶。许多光合生物都利用这种酶来水解叶绿素的植醇尾。虽然传统上认为它催化叶绿素降解的第一步，但最近的研究表明，叶绿素酶反而在非生物胁迫或产生活性氧的条件下发挥作用。然而，关于叶绿素酶在这些条件下如何被调节以发挥功能的分子细节仍然不清楚。在这里，我们使用定点诱变、质谱分析、动态光散射、尺寸排阻多角度光散射以及稳态酶动力学和热稳定性测量等方法，对叶绿素酶同工型CLH2进行了研究。通过这些实验，我们表明CLH2在溶液中以单体形式存在，并且含有两个二硫键。一个二硫键可能位于活性位点，而另一个则将两个N端半胱氨酸残基连接在一起。这些二硫键分别被化学还原剂或化学和基于蛋白质的还原剂裂解，对于维持酶的活性、稳定性和底物范围至关重要。这项工作表明，叶绿素酶中半胱氨酸残基的氧化是控制叶绿素色素水解的一种新出现的调节策略。
