HOIL-1 is an RBR E3 ligase known to ubiquitinate hydroxyl groups found on protein and even sugars. Recent studies have shown that HOIL-1 has activity in glycogen quality control by ubiquitinating maltose and unbranched glucosaccharides [1], and its mutations are closely associated with Polyglucosan Body Myopathy Type 1 (PGBM1), a rare glycogen storage disease causing muscle weakness [2]. However, whether this mutation affects HOIL-1’s sugar ubiquitination activity remains unclear. Here, we curated a list of mutants that decorate the RBR E3 ligase domain, where we cloned, expressed and purified and these mutants for an in vitro ubiquitination assay using heptamaltose as a substrate. Our results demonstrate that frameshift mutations in the catalytic C-terminal H510 completely abolish its activity. Additionally, the E471K mutation significantly reduced the reaction rate, suggesting that the local electrostatic environment maintained by E471 is critical for assisting H510 in deprotonating the sugar hydroxyl group. Lastly, mutation of the IR2 linker also slightly reduces ubiquitination efficiency, highlighting the potential impact of structural flexibility on sugar recognition. These findings provide implications for understanding the molecular mechanism of HOIL-1 in glycogen storage disease.