Purinosomes represent biomolecular condensates that compartmentalize enzymes in the de novo purine synthesis (DNPS) pathway to enhance pathway flux. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remain elusive. We show that the expression of ASB11, a substrate adaptor of Cul5 ubiquitin ligase complex, is elevated by several purinosome-inducing cues, thereby promoting a K6-linked polyubiquitination of the DNPS enzyme PAICS. This ubiquitination facilitates the recruitment of UBPA2, a Ub-binding protein with multiple stretches of intrinsically disordered regions, to confer multivalent interactions for triggering liquid-liquid phase separation in vitro and purinosome assembly in vivo. Under physiological conditions, purinosomes are constitutively formed in cardiomyocytes through ASB11 high expression. Purinosome deficiency in cardiomyocytes causes insufficient purine supply to compromise mtDNA and mtRNA synthesis, thereby impairing mitochondrial dynamics, bioenergetics, and redox homeostasis. Under pathological conditions, ASB11 expression is downregulated in the failing hearts of cardiomyopathy patients and mouse hearts with myocardial infraction. Accordingly, mice with cardiomyocyte-specific Asb11 ablation are vulnerable to cardiac infraction and dysfunctions after ischemia-reperfusion injury. Our study identifies a driving mechanism for purinosome assembly and uncovers the impacts of purinosome formation on mitochondrial functions and heart diseases.