ACS Chem Biol. 2011 Apr 15;6(4):320-4. doi: 10.1021/cb100298r. Epub 2011 Jan 14.
Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of E. coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology and create a foundation for better understanding how post-translational hydroxylation affects proteins.
脯氨酰 4-羟化酶是依赖抗坏血酸的加氧酶，在包括氧感应、siRNA 调节和胶原折叠在内的各种真核生物生物学过程中发挥关键作用。它们通过催化靶蛋白上特定脯氨酸残基的翻译后羟化来发挥功能，形成（2S，4R）-4-羟脯氨酸。到目前为止，这些翻译后修饰的研究受到缺乏用于生产羟化蛋白的原核重组表达系统的限制。通过在异源表达人脯氨酰 4-羟化酶（P4H）的大肠杆菌细胞中引入生物合成旁路来产生抗坏血酸样分子，我们已经创建了一种能够产生高水平（2S，4R）-4-羟脯氨酸的胶原样蛋白的大肠杆菌菌株。使用这个新系统，我们观察到了 P4H 具有连续催化模式的羟化模式，即使没有抗坏血酸，它也是活跃的。我们的结果提供了对 P4H 酶学的深入了解，并为更好地理解翻译后羟化如何影响蛋白质奠定了基础。
