Vitamin B₆ is governed by the local compartmentalization of metabolic enzymes during growth

Vitamin B₆ is a vital micronutrient across cell types and tissues, and dysregulated B₆ levels contribute to human disease. Despite its importance, how B₆ vitamer levels are regulated is not well understood. Here, we provide evidence that B₆ dynamics are rapidly tuned by precise compartmentation of pyridoxal kinase (PDXK), the rate-limiting B₆ enzyme. We show that canonical Wnt rapidly led to the accumulation of inactive B₆ by shunting cytosolic PDXK into lysosomes. PDXK was modified with methyl-arginine Degron (MrDegron), a protein tag for lysosomes, which enabled delivery via microautophagy. Hyperactive lysosomes resulted in the continuous degradation of PDXK and B₆ deficiency that promoted proliferation in Wnt-driven colorectal cancer (CRC) cells. Pharmacological or genetic disruption of the coordinated MrDegron proteolytic pathway was sufficient to reduce CRC survival in cells and organoid models. In sum, this work contributes to the repertoire of micronutrient-regulated processes that enable cancer cell growth and provides insight into the functional impact of B₆ deficiencies for survival.