The precise positioning of catalytic amino acids against the substrate in an enzyme active site is a crucial factor in biocatalysis. Biosynthesis of the chromophores of fluorescent proteins (FPs) is an autocatalytic process that must conform to these requirements. Here, we show that, in addition to the internal amino acid residues in the proximity of the chromophore, chromophore biosynthesis is influenced by the remote amino acids exposed on the outer surface of the β-barrel structure of the FP. It has been shown earlier that chromophore biosynthesis of the red FP from Zoanthus sp. (zoan2RFP) proceeds via an immature green state. At the same time, the green state is the final stage of chromophore biosynthesis of green FP (zoanGFP), which is highly homologous to zoan2RFP. It was also shown that a single N66D substitution in the chromophore-forming sequence of zoanGFP might trigger the synthesis of the red chromophore. However, in this case, the synthesis of the red chromophore is incomplete and occurs only at elevated temperatures. Here, we tried to uncover additional structural determinants that govern the biosynthesis of the red chromophore. A comparison of zoanGFP and zoan2RFP revealed intrabarrel amino acid differences at five positions. Exhaustive substitutions of these five positions in zoanGFP-N66D gave rise to zoanGFPmut with the same intrabarrel amino acid composition as zoan2RFP. zoanGFPmut showed only partial green-to-red chromophore transformation at elevated temperatures. To elucidate the extra factors that can affect red chromophore biosynthesis, we performed comparative molecular dynamics simulations of zoan2RFP and zoanGFPmut. The simulations revealed several external amino acids that might influence the arrangement and flexibility of the chromophore-surrounding amino acid residues in these proteins. Mutagenesis experiments confirmed the crucial role of these residues in red chromophore biosynthesis. The obtained zoanGFPmut2 exhibited complete green-to-red transformation, suggesting that the mutated amino acids exposed on the surface of the β-barrel contribute to red chromophore biosynthesis.

催化氨基酸相对于酶活性位点底物的精确定位是生物催化中的关键因素。荧光蛋白（FPs）发色团的生物合成是一种自动催化过程，必须符合这些要求。在这里，我们表明，除了发色团附近的内部氨基酸残基外，发色团的生物合成还受到暴露于FP的β桶结构外表面的远端氨基酸的影响。早先已经显示，来自火棘的红色FP的生色团生物合成sp。（zoan2RFP）通过未成熟的绿色状态进行。同时，绿色状态是绿色FP（zoanGFP）的生色团生物合成的最后阶段，这与zoan2RFP高度同源。还显示在zoanGFP的生色团形成序列中单个N66D取代可能触发红色生色团的合成。然而，在这种情况下，红色生色团的合成是不完全的，并且仅在升高的温度下发生。在这里，我们试图揭示控制红色生色团生物合成的其他结构决定簇。zoanGFP和zoan2RFP的比较显示了桶内五个位置的氨基酸差异。zoanGFP-N66D中这五个位置的彻底取代产生了zoanGFPmut，其桶内氨基酸组成与zoan2RFP相同。zoanGFPmut在升高的温度下仅显示部分绿色到红色的生色团转化。为了阐明可能影响红色生色团生物合成的其他因素，我们进行了zoan2RFP和zoanGFPmut的比较分子动力学模拟。模拟显示了几种外部氨基酸，这些氨基酸可能会影响这些蛋白质中发色团周围的氨基酸残基的排列和灵活性。诱变实验证实了这些残基在红色生色团生物合成中的关键作用。所获得的zoanGFPmut2表现出完全的绿色到红色的转化，表明暴露在β-桶表面的突变氨基酸有助于红色生色团的生物合成。为了阐明可能影响红色生色团生物合成的其他因素，我们进行了zoan2RFP和zoanGFPmut的比较分子动力学模拟。模拟显示了几种外部氨基酸，这些氨基酸可能会影响这些蛋白质中发色团周围氨基酸残基的排列和灵活性。诱变实验证实了这些残基在红色生色团生物合成中的关键作用。所获得的zoanGFPmut2表现出完全的绿色到红色的转化，表明暴露在β-桶表面的突变氨基酸有助于红色生色团的生物合成。为了阐明可能影响红色生色团生物合成的其他因素，我们进行了zoan2RFP和zoanGFPmut的比较分子动力学模拟。模拟显示了几种外部氨基酸，这些氨基酸可能会影响这些蛋白质中发色团周围的氨基酸残基的排列和灵活性。诱变实验证实了这些残基在红色生色团生物合成中的关键作用。所获得的zoanGFPmut2表现出完全的绿色到红色的转化，表明暴露在β-桶表面的突变氨基酸有助于红色生色团的生物合成。模拟显示了几种外部氨基酸，这些氨基酸可能会影响这些蛋白质中发色团周围的氨基酸残基的排列和灵活性。诱变实验证实了这些残基在红色生色团生物合成中的关键作用。所获得的zoanGFPmut2表现出完全的绿色到红色的转化，表明暴露在β-桶表面的突变氨基酸有助于红色生色团的生物合成。模拟显示了几种外部氨基酸，这些氨基酸可能会影响这些蛋白质中发色团周围的氨基酸残基的排列和灵活性。诱变实验证实了这些残基在红色生色团生物合成中的关键作用。所获得的zoanGFPmut2表现出完全的绿色到红色的转化，表明暴露在β-桶表面的突变氨基酸有助于红色生色团的生物合成。