A mechanism for red coloration in vertebrates

Red coloration is a salient feature of the natural world. Many vertebrates produce red color by converting dietary yellow carotenoids into red ketocarotenoids via an unknown mechanism. Here, we show that two enzymes, cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), are sufficient to catalyze this conversion. In birds, both enzymes are expressed at sites of ketocarotenoid biosynthesis (feather follicles and red cone photoreceptors), and genetic evidence implicates these enzymes in yellow/red color variation in feathers. In fish, homologs of CYP2J19 and BDH1L are required for ketocarotenoid production, and we show that these enzymes are sufficient to produce ketocarotenoids in cell culture and when ectopically expressed in fish skin. Finally, we demonstrate that the red-cone-enriched tetratricopeptide repeat protein 39B (TTC39B) enhances ketocarotenoid production when co-expressed with CYP2J19 and BDH1L. The discovery of this mechanism of ketocarotenoid biosynthesis has major implications for understanding the evolution of color diversity in vertebrates.

Methods
Chromatograms and spectra were collected by reverse-phase high-performance liquid chromatography (HPLC) with UV-Vis DAD detection. Tissue carotenoid content was measured by HPLC. Tissue lipid content was measured with a Miura 200 analyzer with reagent kits (I.S.E. S.r.l.; Guidonia, Italy; A-R0100000901, A-R0200001301, and A-R0200001401).

红色着色是自然界中一种显著的特征。许多脊椎动物通过未知的机制，将膳食来源的黄色类胡萝卜素转化为红色酮类胡萝卜素，以此生成红色体色。本研究证实，细胞色素P450 2J19（cytochrome P450 2J19, CYP2J19）与3-羟基丁酸脱氢酶1样蛋白（3-hydroxybutyrate dehydrogenase 1-like, BDH1L）这两种酶足以催化该转化反应。在鸟类体内，这两种酶均在酮类胡萝卜素的合成位点（羽毛毛囊与红色视锥感光细胞）中表达，且遗传学证据表明它们参与了鸟类羽毛的黄/红色体色变异调控。在鱼类中，CYP2J19与BDH1L的同源蛋白是酮类胡萝卜素生成所必需的，我们的实验证实，在细胞培养体系以及异位表达于鱼类皮肤的情况下，这两种酶均可介导酮类胡萝卜素的产生。最后，我们证实，与CYP2J19和BDH1L共表达时，红色视锥感光细胞富集的四肽重复蛋白39B（tetratricopeptide repeat protein 39B, TTC39B）可增强酮类胡萝卜素的生成效率。这一酮类胡萝卜素生物合成机制的发现，对于理解脊椎动物体色多样性的演化具有重要意义。

实验方法
本研究通过配备紫外-可见二极管阵列（UV-Vis DAD）检测器的反相高效液相色谱（HPLC）采集色谱图与光谱数据。采用HPLC法检测组织中的类胡萝卜素含量。使用Miura 200型分析仪及配套试剂盒（I.S.E. S.r.l.; 意大利吉多尼亚；货号：A-R0100000901、A-R0200001301、A-R0200001401）测定组织脂质含量。