{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T23:16:38Z","timestamp":1789514198655,"version":"build-2803163510"},"reference-count":53,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2022,7,14]],"date-time":"2022-07-14T00:00:00Z","timestamp":1657756800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"National Science Centre (NCN)","doi-asserted-by":"publisher","award":["UMO-2020\/39\/B\/NZ7\/01519"],"award-info":[{"award-number":["UMO-2020\/39\/B\/NZ7\/01519"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Due to the apparent similarity of fungal and mammalian metabolic pathways, the number of established antifungal targets is low, and the identification of novel ones is highly desirable. The results of our studies, presented in this work, indicate that the fungal biosynthetic pathway of L-methionine, an amino acid essential for humans, seems to be an attractive perspective. The MET2 gene from Candida albicans encoding L-homoserine O-acetyltransferase (CaMet2p), an enzyme catalyzing the first step in that pathway, was cloned and expressed as the native or the oligo-His-tagged fusion protein in Escherichia coli. The recombinant enzymes were purified and characterized for their basic molecular properties and substrate specificities. The purified MET2 gene product revealed the appropriate activity, catalyzed the conversion of L-homoserine (L-Hom) to O-acetyl-L-homoserine (OALH), and exhibited differential sensitivity to several L-Hom or OALH analogues, including penicillamine. Surprisingly, both penicillamine enantiomers (L- and D-Pen) displayed comparable inhibitory effects. The results of the docking of L- and D-Pen to the model of CaMet2p confirmed that both enantiomeric forms of the inhibitor are able to bind to the catalytic site of the enzyme with similar affinities and a similar binding mode. The sensitivity of some fungal cells to L-Pen, depending on the presence or absence of L-Met in the medium, clearly indicate Met2p targeting. Moreover, C. glabrata clinical strains that are resistant to fluconazole displayed a similar susceptibility to L-Pen as the wild-type strains. Our results prove the potential usefulness of Met2p as a molecular target for antifungal chemotherapy.<\/jats:p>","DOI":"10.3390\/ijms23147763","type":"journal-article","created":{"date-parts":[[2022,7,15]],"date-time":"2022-07-15T00:17:10Z","timestamp":1657844230000},"page":"7763","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Antifungal Effect of Penicillamine Due to the Selective Targeting of L-Homoserine O-Acetyltransferase"],"prefix":"10.3390","volume":"23","author":[{"given":"Aleksandra","family":"Kupli\u0144ska","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kamila","family":"Rz\u0105d","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3003-8483","authenticated-orcid":false,"given":"Marek","family":"Wojciechowski","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2616-4495","authenticated-orcid":false,"given":"S\u0142awomir","family":"Milewski","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0833-5596","authenticated-orcid":false,"given":"Iwona","family":"Gabriel","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, 80-233 Gdansk, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1126\/scitranslmed.3004404","article-title":"Hidden killers: Human fungal infections","volume":"4","author":"Brown","year":"2012","journal-title":"Sci. Transl. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1013","DOI":"10.1016\/S0140-6736(05)67381-3","article-title":"Advances and challenges in management of invasive mycoses","volume":"366","author":"Patterson","year":"2005","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"214","DOI":"10.3389\/fmicb.2019.00214","article-title":"The still underestimated problem of fungal diseases worldwide","volume":"10","author":"Almeida","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1016\/j.ijantimicag.2017.05.019","article-title":"Epidemiology of antifungal resistance in human pathogenic yeasts: Current viewpoint and practical recommendations for management","volume":"50","author":"Farmakiotis","year":"2017","journal-title":"Int. J. Antimicrob. Agents"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/mBio.02048-18","article-title":"Gene essentiality analyzed by in vivo transposon mutagenesis and machine learning in a stable haploid isolate of candida albicans","volume":"9","author":"Segal","year":"2018","journal-title":"MBio"},{"key":"ref_6","first-page":"3","article-title":"Molecular targets for antifungals in amino acid and protein biosynthetic pathways","volume":"1","year":"2021","journal-title":"Amino Acids"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.fgb.2017.11.009","article-title":"Versatility of putative aromatic aminotransferases from Candida albicans","volume":"110","author":"Milewski","year":"2018","journal-title":"Fungal Genet. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1093\/ajcn\/53.3.670","article-title":"Plasma amino acid concentrations and amino acid ratios in normal adults and adults heterozygous for phenylketonuria ingesting a hamburger and milk shake meal","volume":"53","author":"Stegink","year":"1991","journal-title":"Am. J. Clin. Nutr."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.1128\/AAC.01400-06","article-title":"Role of homoserine transacetylase as a new target for antifungal agents","volume":"51","author":"Nazi","year":"2007","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1099\/mic.0.034348-0","article-title":"Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2\u0394) mutants is influenced by the carbon source and rapamycin","volume":"156","author":"Kingsbury","year":"2010","journal-title":"Microbiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1128\/jb.118.3.911-918.1974","article-title":"Characteristics and relationships of mercury resistant mutants and methionine auxotrophs of yeast","volume":"118","author":"Singh","year":"1974","journal-title":"J. Bacteriol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1002\/yea.3012","article-title":"Disrupting the methionine biosynthetic pathway in Candida guilliermondii: Characterization of the MET2 gene as counter-selectable marker","volume":"31","author":"Blanc","year":"2014","journal-title":"Yeast"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"137","DOI":"10.26650\/IstanbulJPharm.2020.0004","article-title":"Fungal homoserine transacetylase: A potential antifungal target","volume":"51","author":"Seyran","year":"2021","journal-title":"\u0130stanb. J. Pharm."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10008","DOI":"10.1073\/pnas.1513033112","article-title":"Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis","volume":"112","author":"Berney","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-019-56722-2","article-title":"Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site","volume":"9","author":"Chaton","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"D592","DOI":"10.1093\/nar\/gkw924","article-title":"The Candida Genome Database (CGD): Incorporation of Assembly 22, systematic identifiers and visualization of high throughput sequencing data","volume":"45","author":"Skrzypek","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D., and Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook, Humana Press.","DOI":"10.1385\/1-59259-890-0:571"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"13560","DOI":"10.1021\/bi0514764","article-title":"Catalytic mechanism of fungal homoserine transacetylase","volume":"44","author":"Nazi","year":"2005","journal-title":"Biochemistry"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript server","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"15768","DOI":"10.1021\/bi051951y","article-title":"Crystal structure of homoserine transacetylase from Haemophilus influenzae reveals a new family of \u03b1\/\u03b2-hydrolases","volume":"44","author":"Mirza","year":"2005","journal-title":"Biochemistry"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1093\/bioinformatics\/14.1.48","article-title":"Combining evidence using p-values: Application to sequence homology searches","volume":"14","author":"Bailey","year":"1998","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"R24","DOI":"10.1186\/gb-2007-8-2-r24","article-title":"Quantifying similarity between motifs","volume":"8","author":"Gupta","year":"2007","journal-title":"Genome Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1093\/bioinformatics\/btr064","article-title":"FIMO: Scanning for occurrences of a given motif","volume":"27","author":"Grant","year":"2011","journal-title":"Bioinformatics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3897","DOI":"10.1016\/S0021-9258(19)41483-X","article-title":"Purification and properties of homoserine transacetylase from Bacillus polymyxa","volume":"250","author":"Wyman","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1493","DOI":"10.1093\/oxfordjournals.jbchem.a133342","article-title":"Feedback inhibition by methionine and S-adenosylmethionine, and desensitization of homoserine O-acetyltransferase in Brevibacterium flavum","volume":"89","author":"Shiio","year":"1981","journal-title":"J. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3458","DOI":"10.1128\/jb.169.8.3458-3463.1987","article-title":"Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae","volume":"169","author":"Yamagata","year":"1987","journal-title":"J. Bacteriol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1016\/j.bbrc.2019.07.117","article-title":"Crystal structure and biochemical characterization of O-acetylhomoserine acetyltransferase from Mycobacterium smegmatis ATCC 19420","volume":"517","author":"Sagong","year":"2019","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1039\/c2md20020a","article-title":"L-Penicillamine is a mechanism-based inhibitor of serine palmitoyltransferase by forming a pyridoxal-5\u2032-phosphate-thiazolidine adduct","volume":"3","author":"Lowther","year":"2012","journal-title":"Medchemcomm"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1028","DOI":"10.1016\/j.cgh.2013.03.012","article-title":"Efficacy and safety of oral chelators in treatment of patients with wilson disease","volume":"11","author":"Weiss","year":"2013","journal-title":"Clin. Gastroenterol. Hepatol."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Eidelman, C., and Lowry, J.A. (2016). D-Penicillamine. Critical Care Toxicology, Springer International Publishing.","DOI":"10.1007\/978-3-319-20790-2_182-1"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Borzyszkowska-Bukowska, J., G\u00f3rska, J., Szczeblewski, P., Laskowski, T., Gabriel, I., Jurasz, J., Koz\u0142owska-Tylingo, K., Szweda, P., and Milewski, S. (2021). Quest for the molecular basis of improved selective toxicity of all-trans isomers of aromatic heptaene macrolide antifungal antibiotics. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms221810108"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fmicb.2019.02853","article-title":"The synergistic antifungal effect and potential mechanism of D-Penicillamine combined with fluconazole against Candida albicans","volume":"10","author":"Li","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1146\/annurev.micro.51.1.73","article-title":"Molecular genetics of sulfur assimilation in filamentous fungi and yeast","volume":"51","author":"Marzluf","year":"1997","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1111\/j.1567-1364.2011.00725.x","article-title":"Biodiversity in sulfur metabolism in hemiascomycetous yeasts","volume":"11","author":"Casaregola","year":"2011","journal-title":"FEMS Yeast Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1093\/genetics\/130.1.51","article-title":"Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: Updating of the sulfur metabolism pathway","volume":"130","author":"Cherest","year":"1992","journal-title":"Genetics"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"19714","DOI":"10.1074\/jbc.M111.240648","article-title":"CgCYN1, a plasma membrane cystine-specific transporter of Candida glabrata with orthologues prevalent among pathogenic yeast and fungi","volume":"286","author":"Yadav","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1016\/S0022-2836(05)80360-2","article-title":"Basic local alignment search tool","volume":"215","author":"Altschul","year":"1990","journal-title":"J. Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1562","DOI":"10.1128\/EC.4.9.1562-1573.2005","article-title":"Genome-wide transcription profiling of the early phase of biofilm formation by Candida albicans","volume":"4","author":"Murillo","year":"2005","journal-title":"Eukaryot. Cell"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1111\/j.1432-1033.1990.tb15542.x","article-title":"GAP1, the general amino acid permease gene of Saccharomyces cerevisiae Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression","volume":"190","author":"Jauniaux","year":"1990","journal-title":"Eur. J. Biochem."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1007\/s002940050506","article-title":"Substrate specificity and gene expression of the amino acid permeases in Saccharomyces cerevisiae","volume":"36","author":"Regenberg","year":"1999","journal-title":"Curr. Genet."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Martho, K.F.C., De Melo, A.T., Takahashi, J.P.F., Guerra, J.M., Da Silva Santos, D.C., Purisco, S.U., Melhem, M.D.S.C., Dos Anjos Fazioli, R., Phanord, C., and Sartorelli, P. (2016). Amino acid permeases and virulence in Cryptococcus neoformans. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0163919"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1159\/000466688","article-title":"Serum amino acid profiles in normal subjects and in patients with or at risk of Alzheimer dementia","volume":"7","author":"Corso","year":"2017","journal-title":"Dement. Geriatr. Cogn. Dis. Extra"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1038\/ejcn.2015.144","article-title":"Plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: A cross-sectional analysis in the EPIC-Oxford cohort","volume":"70","author":"Schmidt","year":"2016","journal-title":"Eur. J. Clin. Nutr."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1904","DOI":"10.1016\/j.transproceed.2012.11.028","article-title":"Higher serum methionine levels as a predictive factor in patients with irreversible fulminant hepatic failure","volume":"45","author":"Sato","year":"2013","journal-title":"Transplant. Proc."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"610","DOI":"10.1099\/jmm.0.000062","article-title":"Mechanisms of azole resistance among clinical isolates of Candida glabrata in Poland","volume":"64","author":"Szweda","year":"2015","journal-title":"J. Med. Microbiol."},{"key":"ref_47","unstructured":"Lazar, I., and Lazar, I. (2022, January 10). GelAnalyzer 19.1. Available online: www.gelanalyzer.com."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1007\/978-1-4939-6850-3_2","article-title":"DTNB-based quantification of in vitro enzymatic N-terminal acetyltransferase activity","volume":"Volume 1574","author":"Schilling","year":"2017","journal-title":"Methods in Molecular Biology"},{"key":"ref_49","first-page":"M27-A3","article-title":"Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved standar\u2014Third Edition","volume":"28","author":"Wayne","year":"2008","journal-title":"CLSI Doc."},{"key":"ref_50","first-page":"1010","article-title":"HyperChem: A software package for computational chemistry and molecular modeling","volume":"14","author":"Froimowitz","year":"1993","journal-title":"Biotechniques"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2785","DOI":"10.1002\/jcc.21256","article-title":"Software news and updates AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility","volume":"30","author":"Morris","year":"2009","journal-title":"J. Comput. Chem."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.jmgm.2017.09.016","article-title":"Simplified AutoDock force field for hydrated binding sites","volume":"78","author":"Wojciechowski","year":"2017","journal-title":"J. Mol. Graph. Model."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7763\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:50:11Z","timestamp":1760140211000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/14\/7763"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,14]]},"references-count":53,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["ijms23147763"],"URL":"https:\/\/doi.org\/10.3390\/ijms23147763","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,14]]}}}