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More than 300,000 site-specific phosphorylation sites have been detected across\u2009&gt;\u200920,000 human proteins, yet only a small fraction (~\u20095%) have been experimentally characterized, and their roles in health and disease remain largely unexplored. In particular, the functional consequences of most phosphorylation events in metabolic enzymes remain unknown. Here, we investigated the functional impact of modifying Ser81 in alanine:glyoxylate aminotransferase (AGT), the key enzyme responsible for glyoxylate detoxification and whose loss-of-function causes Primary Hyperoxaluria Type I (PH1). We examined phosphomimetic substitutions at Ser81 in the WT (wild-type) enzyme, the common polymorphic minor allele (LM, containing two variations, p.P11L and p.I340M), and the two most frequent PH1-associated variants (LM-p.G170R and LM-p.I244T). Using biochemical, biophysical and cell-based approaches, we found that introducing a negative charge at Ser81 (through the S81D substitution) strongly perturbs PLP (pyridoxal 5\u00b4-phosphate)\/PMP (pyridoxamine 5\u00b4-phosphate) binding pose and disrupts catalytic activity, while preserving secondary and tertiary structure as well as peroxisomal localization. In contrast, the non-charged S81A substitution produced milder effects. These results indicate that Ser81 contributes to stabilizing the cofactor interaction network at the AGT active site. Our findings therefore identify Ser81 as a previously uncharacterized regulatory position that can critically influence AGT activity. Although further work is required to determine the physiological frequency and regulatory context of this modification in vivo, our results suggest that phosphorylation at this position could represent an additional modulatory layer influencing AGT function and genotype\u2013phenotype relationships in PH1, with potential implications for understanding regulatory mechanisms affecting AGT activity in disease.<\/jats:p>","DOI":"10.1186\/s43556-026-00442-3","type":"journal-article","created":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T06:49:56Z","timestamp":1776149396000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Impact of Ser81 phosphorylation on alanine: glyoxylate aminotransferase associated with Primary hyperoxaluria type I"],"prefix":"10.1186","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-8214-2008","authenticated-orcid":false,"given":"Sara","family":"Milosevic","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9599-9854","authenticated-orcid":false,"given":"Eduardo","family":"Salido","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6653-6260","authenticated-orcid":false,"given":"Noel","family":"Mesa-Torres","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7706-3243","authenticated-orcid":false,"given":"Angel L.","family":"Pey","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0627-3379","authenticated-orcid":false,"given":"Mario","family":"Cano-Mu\u00f1oz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"442_CR1","doi-asserted-by":"publisher","unstructured":"Neira JL, Rizzuti B, Jim\u00e9nez-Alesanco A, Palomino-Sch\u00e4tzlein M, Abi\u00e1n O, Vel\u00e1zquez-Campoy A, et al. 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A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation. FEBS Lett. 2022. https:\/\/doi.org\/10.1002\/1873-3468.14238.","journal-title":"FEBS Lett"},{"key":"442_CR7","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2020.06.043","author":"G P\u00e9rez-Mej\u00edas","year":"2020","unstructured":"P\u00e9rez-Mej\u00edas G, Vel\u00e1zquez-Cruz A, Guerra-Castellano A, Ba\u00f1os-Jaime B, D\u00edaz-Quintana A, Gonz\u00e1lez-Arzola K, et al. Exploring protein phosphorylation by combining computational approaches and biochemical methods. Comput Struct Biotechnol J. 2020. https:\/\/doi.org\/10.1016\/j.csbj.2020.06.043.","journal-title":"Comput Struct Biotechnol J"},{"key":"442_CR8","doi-asserted-by":"publisher","DOI":"10.1038\/nature13999","author":"A Bah","year":"2015","unstructured":"Bah A, Vernon RM, Siddiqui Z, Krzeminski M, Muhandiram R, Zhao C, et al. 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Chemistry. 2015. https:\/\/doi.org\/10.1002\/chem.201502019.","journal-title":"Chemistry"},{"key":"442_CR23","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0071963","author":"N Mesa-Torres","year":"2013","unstructured":"Mesa-Torres N, Fabelo-Rosa I, Riverol D, Yunta C, Albert A, Salido E, et al. The role of protein denaturation energetics and molecular chaperones in the aggregation and mistargeting of mutants causing primary hyperoxaluria type I. PLoS ONE. 2013. https:\/\/doi.org\/10.1371\/journal.pone.0071963.","journal-title":"PLoS ONE"},{"key":"442_CR24","doi-asserted-by":"publisher","DOI":"10.1007\/s00726-010-0801-2","author":"AL Pey","year":"2011","unstructured":"Pey AL, Salido E, Sanchez-Ruiz JM. Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria. 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