Trichoderma virens genome harbors two isoforms of GAPDH, one (gGPD) involved in glycolysis and the other one (vGPD) in secondary metabolism. vGPD is expressed as part of the “vir” cluster responsible for the biosynthesis of volatile sesquiterpenes. The secondary metabolism-associated GAPDH is tolerant to the anti-cancer metabolite heptelidic acid (HA), produced by T. virens. Characterizing the HA-tolerant form of GAPDH, thus has implications in cancer therapy. In order to get insight into the mechanism of HA-tolerance of vGPD, we have purified recombinant form of this protein. The protein displays biochemical and biophysical characteristics analogous to the gGPD isoform. It exists as a tetramer with Tm of about 56.5 °C, and displays phosphorylation enzyme activity with Km and Kcat of 0.38 mM and 2.55 sec⁻¹, respectively. The protein weakly binds to the sequence upstream of the vir4 gene that codes for the core enzyme (a terpene cyclase) of the “vir” cluster. The EMSA analysis indicates that vGPD may not act as a transcription factor driving the “vir” cluster, at least not by directly binding to the promoter region. We also succeeded in obtaining small crystals of this protein. We have constructed structural models of vGPD and gGPD of T. virens. In silico constrained docking analysis reveals weaker binding of heptelidic acid in vGPD, compared to gGPD protein.

里氏木霉基因组具有两种GAPDH亚型，一种（gGPD）参与糖酵解，另一种（vGPD）参与次级代谢。vGPD表示为负责挥发性倍半萜生物合成的“ vir”簇的一部分。次级代谢相关GAPDH耐受抗癌代谢物heptelidic酸（HA），通过产生的绿木霉。因此，表征GA耐受HA的GAPDH形式对癌症治疗具有重要意义。为了深入了解vGPD的HA耐受机制，我们纯化了该蛋白的重组形式。该蛋白质具有类似于gGPD同工型的生化和生物物理特征。它以四聚体的形式存在，其T m约为56.5°C，并显示磷酸化酶的活性。ķ m和ķ 0.38 mM和2.55秒的猫^-1分别。该蛋白与vir 4基因上游序列弱结合，该序列编码“ vir”簇的核心酶（萜烯环化酶）。EMSA分析表明，vGPD可能不充当驱动“ vir”簇的转录因子，至少不能直接结合启动子区域。我们还成功获得了这种蛋白质的小晶体。我们已经构建VGPD和gGPD的结构模型绿木霉。在计算机上受约束的对接分析显示，与gGPD蛋白相比，vGPD中的庚二酸结合更弱。