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Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3\u0394 mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with Khalf values of 65.5 \u00b1 7.11 \u00b5M for putrescine and 66.9 \u00b1 2.09 \u00b5M for dc-SAM, and Vmax values of 7.1 \u00b1 0.57 and 7.9 \u00b1 0.12 nmol\u00b7\u00b5g\u22121\u00b7min\u22121, respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase.<\/jats:p>","DOI":"10.3390\/pathogens15040432","type":"journal-article","created":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T14:05:36Z","timestamp":1776348336000},"page":"432","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional and Biochemical Characterization of Spermidine Synthase CauSpe3 from Candidozyma auris"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9853-0987","authenticated-orcid":false,"given":"Jae-Yeon","family":"Choi","sequence":"first","affiliation":[{"name":"Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2318-5960","authenticated-orcid":false,"given":"Pallavi","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Choukri","family":"Ben Mamoun","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06520, USA"},{"name":"Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Chowdhary, A., Sharma, C., and Meis, J.F. 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