Antimicrobial resistance is an emerging threat for public health. The success of resistance mutations depends on the trade-off between the benefits and costs they incur. This trade-off is largely unknown and uncharacterized for antifungals. Here, we systematically measure the effect of all amino acid substitutions in the yeast cytosine deaminase Fcy1, the target of the antifungal 5-fluorocytosine (5-FC, flucytosine). We identify over 900 missense mutations granting resistance to 5-FC, a large fraction of which appear to act through destabilization of the protein. The relationship between 5-FC resistance and growth sustained by cytosine deamination is characterized by a sharp trade-off, such that small gains in resistance universally lead to large losses in canonical enzyme function. We show that this steep relationship can be explained by differences in the dose–response functions of 5-FC and cytosine. Finally, we observe the same trade-off shape for the orthologue of FCY1 in Cryptoccocus neoformans, a human pathogen. Our results provide a powerful resource and platform for interpreting drug target variants in fungal pathogens as well as unprecedented insights into resistance–function trade-offs.

抗菌素耐药性是公共卫生的新威胁。抗性突变的成功取决于它们产生的收益和成本之间的权衡。对于抗真菌剂，这种权衡在很大程度上是未知的和没有特征的。在这里，我们系统地测量了酵母胞嘧啶脱氨酶 Fcy1 中所有氨基酸取代的影响，这是抗真菌剂 5-氟胞嘧啶（5-FC，氟胞嘧啶）的靶标。我们确定了超过 900 种赋予 5-FC 抗性的错义突变，其中很大一部分似乎是通过蛋白质的不稳定而起作用。5-FC 抗性与胞嘧啶脱氨作用所维持的生长之间的关系以急剧权衡为特征，因此抗性的小幅增加通常会导致经典酶功能的大量损失。我们表明，这种陡峭的关系可以通过 5-FC 和胞嘧啶的剂量反应函数的差异来解释。最后，我们观察到相同的折衷形状新型隐球菌中的FCY1，一种人类病原体。我们的结果为解释真菌病原体中的药物靶标变异提供了强大的资源和平台，并为耐药性功能权衡提供了前所未有的见解。