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Although there is agreement in the literature that CBS is a homomultimer, its precise structure is a source of confusion. Here, we performed a series of experiments examining the quaternary structure of various wild\u2010type and mutant CBS enzymes using a combination of native gel electrophoresis, in situ activity assays, analytical ultracentrifugation, and gel filtration. Our data show that recombinantly expressed and purified full\u2010length wild\u2010type human CBS enzyme (hCBS) and HCU\u2010causing variants (p.P422L, p.I435T, and p.R125Q CBS) form high molecular weight assemblies that are consistent with the properties expected of a filament. The filament is enzymatically active, and its size is sensitive to protein concentration. This behavior contrasts sharply with hCBS enzymes containing small deletions within the Bateman domain, which form stable tetramers and octamers regardless of concentration. Examination of liver lysates from humans and mice confirms the existence of enzymatically active high molecular weight aggregates in vivo, but also shows that these aggregates are specific to human CBS and do not occur in mice. Molecular modeling using AlphaFold2 suggests that these experimentally observed differences may be explained by subtle differences in the interaction mediated by the Bateman domains. Our results show that small differences in amino acid sequence can cause large differences in the size and shape of CBS multimers.<\/jats:p>","DOI":"10.1002\/pro.5223","type":"journal-article","created":{"date-parts":[[2024,11,16]],"date-time":"2024-11-16T10:18:43Z","timestamp":1731752323000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Impact of primary sequence changes on the self\u2010association properties of mammalian cystathionine beta\u2010synthase enzymes"],"prefix":"10.1002","volume":"33","author":[{"given":"Hyung\u2010Ok","family":"Lee","sequence":"first","affiliation":[{"name":"Cancer Signaling and Microenvironment Program Fox Chase Cancer Center  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kushol","family":"Gupta","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics University of Pennsylvania  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liqun","family":"Wang","sequence":"additional","affiliation":[{"name":"Cancer Signaling and Microenvironment Program Fox Chase Cancer Center  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roland L.","family":"Dunbrack","sequence":"additional","affiliation":[{"name":"Cancer Signaling and Microenvironment Program Fox Chase Cancer Center  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2751-2124","authenticated-orcid":false,"given":"Tomas","family":"Majtan","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Faculty of Science and Medicine University of Fribourg  Fribourg Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4990-3695","authenticated-orcid":false,"given":"Warren 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