MINDY-1 is a recently discovered new family of deubiquitinating enzymes (DUB), but one of its yeast homologs, YGL082W, does not show any DUB activity in vitro. Sequence alignment shows that YGL082W possesses the correct catalytic triad, and yet did not catalyze either the hydrolysis of di-ubiquitin, crosslinking with C-terminally propargylated ubiquitin, or hydrolysis of ubiquitin-7-amino-4-methylcoumarin. After obtaining a crystal structure of the catalytic domain of YGL082W, we identified an interesting difference between the catalytic center loop of YGL082W and that of its human homolog MINDY-1. Because the conformation of the catalytic center loop was previously reported to be important for the deubiquitination activity of MINDY-1, we hypothesized that Glu27 (instead of the corresponding Pro136 in MINDY-1) of the catalytic center loop of YGL082W may impair the conformational change and account for the lack of activity. This hypothesis was supported by homology modeling and molecular dynamics simulations, which showed that the Pro-to-Glu mutation (P136E mutation for MINDY-1) creates a hydrogen bond that inhibits the conformation change of the catalytic center loop of MINDY-1. Further experiments through site-directed mutation validated this hypothesis, showing that the P27E mutation caused MIY1 (a homologous active DUB from yeast) to lose activity.

中文翻译：

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YGL082W的体外失活是由于催化中心环的构象变化受到损害

MINDY-1是最近发现的新的去泛素化酶（DUB）家族，但其酵母同源物之一YGL082W在体外未显示任何DUB活性。。序列比对显示，YGL082W具有正确的催化三联体，但既不催化双泛素的水解，与C末端炔丙基化的泛素的交联，也不催化泛素7-氨基-4-甲基香豆素的水解。获得YGL082W催化结构域的晶体结构后，我们发现YGL082W的催化中心环与其人类同源物MINDY-1的催化中心环之间存在有趣的区别。由于以前报道催化中心环的构象对于MINDY-1的去泛素化活性很重要，我们假设YGL082W催化中心环的Glu27（而不是MINDY-1中的相应Pro136）可能会损害构象变化并说明缺乏活动。同源性模型和分子动力学模拟支持该假设，表明Pro-to-Glu突变（MINDY-1的P136E突变）产生氢键，该氢键抑制MINDY-1催化中心环的构象变化。通过定点突变进行的进一步实验验证了这一假设，表明P27E突变导致MIY1（来自酵母的同源活性DUB）丧失活性。