{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T10:09:20Z","timestamp":1773828560349,"version":"3.50.1"},"reference-count":49,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,2,12]],"date-time":"2020-02-12T00:00:00Z","timestamp":1581465600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007029","name":"State Committee of Science","doi-asserted-by":"publisher","award":["13-2I344"],"award-info":[{"award-number":["13-2I344"]}],"id":[{"id":"10.13039\/501100007029","id-type":"DOI","asserted-by":"publisher"}]},{"name":"State Committee of Science of the Ministry of Education and Science of the Republic of Armenia","award":["no. 13-2I344"],"award-info":[{"award-number":["no. 13-2I344"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["High-Throughput"],"abstract":"<jats:p>D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.<\/jats:p>","DOI":"10.3390\/ht9010005","type":"journal-article","created":{"date-parts":[[2020,2,14]],"date-time":"2020-02-14T10:09:57Z","timestamp":1581674997000},"page":"5","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)"],"prefix":"10.3390","volume":"9","author":[{"given":"Hovsep","family":"Aganyants","sequence":"first","affiliation":[{"name":"SPC Armbiotechnology, National Academy of Sciences of Republic of Armenia, Yerevan 0056, Armenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2952-2748","authenticated-orcid":false,"given":"Pierre","family":"Weigel","sequence":"additional","affiliation":[{"name":"UFIP CNRS 6286, Facult\u00e9 de Biologie, Universit\u00e9 de Nantes, 44322 Nantes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yeranuhi","family":"Hovhannisyan","sequence":"additional","affiliation":[{"name":"SPC Armbiotechnology, National Academy of Sciences of Republic of Armenia, Yerevan 0056, Armenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mich\u00e8le","family":"Lecocq","sequence":"additional","affiliation":[{"name":"IICiMed, Facult\u00e9 de Pharmacie, Universit\u00e9 de Nantes, 44035 Nantes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haykanush","family":"Koloyan","sequence":"additional","affiliation":[{"name":"SPC Armbiotechnology, National Academy of Sciences of Republic of Armenia, Yerevan 0056, Armenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9526-7768","authenticated-orcid":false,"given":"Artur","family":"Hambardzumyan","sequence":"additional","affiliation":[{"name":"SPC Armbiotechnology, National Academy of Sciences of Republic of Armenia, Yerevan 0056, Armenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anichka","family":"Hovsepyan","sequence":"additional","affiliation":[{"name":"SPC Armbiotechnology, National Academy of Sciences of Republic of Armenia, Yerevan 0056, Armenia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jean-No\u00ebl","family":"Hallet","sequence":"additional","affiliation":[{"name":"UFIP CNRS 6286, Facult\u00e9 de Biologie, Universit\u00e9 de Nantes, 44322 Nantes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0513-8434","authenticated-orcid":false,"given":"Vehary","family":"Sakanyan","sequence":"additional","affiliation":[{"name":"IICiMed, Facult\u00e9 de Pharmacie, Universit\u00e9 de Nantes, 44035 Nantes, France"},{"name":"IICiMed, Facult\u00e9 de Biologie, Universit\u00e9 de Nantes, 44332 Nantes, France"},{"name":"ProtNeteomix, 44332 Nantes, France"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,2,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1128\/br.40.2.403-468.1976","article-title":"Degradation of purines and pyrimidines by micro-organisms","volume":"40","author":"Vogels","year":"1976","journal-title":"Bacteriol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1002\/(SICI)1097-0134(199705)28:1<72::AID-PROT7>3.0.CO;2-L","article-title":"An evolutionary treasure: Unification of a broad set of amidohydrolases related to urease","volume":"28","author":"Holm","year":"1997","journal-title":"Proteins"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1007\/s00239-013-9580-1","article-title":"Evolution of cyclic amidohydrolases: A highly diversified superfamily","volume":"77","author":"Barba","year":"2013","journal-title":"J. Mol. Evol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/0014-5793(75)80714-9","article-title":"Substrate and steric specificity of hydropyrimidine hydrase","volume":"57","author":"Cecere","year":"1975","journal-title":"FEBS Lett."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1007\/s002530051395","article-title":"Microbial hydantoinases - industrial enzymes from the origin of life?","volume":"51","author":"Syldatk","year":"1999","journal-title":"Appl. Microb. Biotechnol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.1007\/s00253-011-3691-7","article-title":"Stereoselective hydrolysis of aryl-substituted dihydropyrimidines by hydantoinases","volume":"94","author":"Engel","year":"2012","journal-title":"Appl. Microb. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"564","DOI":"10.1021\/bp0000611","article-title":"Production of D-Amino Acid Using Whole Cells of Recombinant Escherichia coli with Separately and Coexpressed D-Hydantoinase and N-Carbamoylase","volume":"16","author":"Park","year":"2000","journal-title":"Biotechnol. Prog."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.mimet.2015.02.002","article-title":"Genetic engineering of Geobacillus spp.","volume":"111","year":"2015","journal-title":"J. Microbiol. Methods"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1016\/j.synbio.2018.05.002","article-title":"An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bottom-uppathway design","volume":"3","author":"Shi","year":"2018","journal-title":"Synth. Syst. Biotechnol."},{"key":"ref_10","first-page":"553","article-title":"Hydantoinase and carbamoylase of thermophilic bacteria","volume":"2","author":"Sakanyan","year":"1998","journal-title":"Recent. Res. Dev. Microb."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1621","DOI":"10.1271\/bbb.58.1621","article-title":"A thermostable hydantoinase of Bacillus stearothermophilus NS1122A: Cloning, sequencing, and high expression of the enzyme gene, and some properties of the expressed enzyme","volume":"58","author":"Mukohara","year":"1994","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1007\/BF02788001","article-title":"Purification and characterization of thermostable D-hydantoinase from thermophilic Bacillus stearothermophilus SD1","volume":"62","author":"Lee","year":"1997","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1385\/ABAB:81:1:53","article-title":"Purification and characterization of thermostable D-hydantoinase from Bacillus thermocatenulatus GH-2","volume":"81","author":"Park","year":"1999","journal-title":"Appl. Biochem. Biothecnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"9410","DOI":"10.1021\/bi0201567","article-title":"Crystal structure of D-hydantoinase from Bacillus stearothermophilus: Insight into the stereochemistry of enantioselectivity","volume":"41","author":"Cheon","year":"2002","journal-title":"Biochemistry"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.jprot.2013.08.012","article-title":"Beyond of gene expression: The impact of protein post-translational modifications in bacteria","volume":"97","author":"Cain","year":"2014","journal-title":"J. Proteom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7413","DOI":"10.1021\/bi036330o","article-title":"Manipulation of the active site loops of D-hydantoinase, a (beta\/alpha)8-barrel protein, for modulation of the substrate specificity","volume":"43","author":"Cheon","year":"2004","journal-title":"Biochemistry"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1007\/s12010-007-8004-3","article-title":"The flexibility of the non-conservative region at the C terminus of D-hydantoinase from Pseudomonas putida YZ-26 is extremely limited","volume":"144","author":"Zhang","year":"2008","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/S0022-2836(02)00422-9","article-title":"X-ray structure of a dihydropyrimidinase from Thermus sp. at 1.3 \u00c5 resolution","volume":"320","author":"Abendroth","year":"2002","journal-title":"J. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"7021","DOI":"10.1128\/JB.182.24.7021-7028.2000","article-title":"Functional expression and characterization of the two cyclic amidohydrolase enzymes, allantoinase and a novel phenylhydantoinase, from Escherichia coli","volume":"182","author":"Kim","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.enzmictec.2008.10.020","article-title":"Designing the substrate specificity of D-hydantoinase using a rational approach","volume":"44","author":"Lee","year":"2009","journal-title":"Enzym. Microb. Technol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1451","DOI":"10.1021\/acs.biochem.8b00896","article-title":"In Silico Protein Design Promotes the Rapid Evolution of Industrial Enzymes","volume":"58","author":"Wang","year":"2019","journal-title":"Biochemistry"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1021\/cr60145a001","article-title":"The Chemistry of Hydantoins","volume":"46","author":"Ware","year":"1950","journal-title":"Chem. Rev."},{"key":"ref_23","first-page":"91","article-title":"Obtention d\u2019amino acides optiquement actifs \u00e0 l\u2019aide d\u2019hydantoinases","volume":"1\u20132","author":"Guivarch","year":"1980","journal-title":"Bull. Soc. Chim."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/S0076-6879(83)91014-5","article-title":"Determination of total protein","volume":"91","author":"Peterson","year":"1983","journal-title":"Meth. Enzym."},{"key":"ref_25","unstructured":"Cornish-Bowden, E. (1979). Fundamentals of Enzyme Kinetics, Medicina. (In Russian)."},{"key":"ref_26","first-page":"6","article-title":"Statistical analyses of enzyme kinetics: Simple Michaelis-Menten and bi-bi ping-pong","volume":"69","author":"Hambardzumyan","year":"2017","journal-title":"Biol. J. Armen."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"S162","DOI":"10.1002\/elps.200900140","article-title":"Automated comparative protein structure modeling with SWISS-MODEL and Swiss-Pdb Viewer: A historical perspective","volume":"30","author":"Guex","year":"2009","journal-title":"Electrophoresis"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"W407","DOI":"10.1093\/nar\/gkm290","article-title":"ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins","volume":"35","author":"Wiederstein","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDockVina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1186\/1475-2859-7-34","article-title":"Engineering inclusion bodies for nondenaturing extraction of functional proteins","volume":"7","author":"Peternel","year":"2008","journal-title":"Microb. Cell Fact."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3878","DOI":"10.1128\/aem.59.11.3878-3888.1993","article-title":"Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus Stearothermophilus","volume":"59","author":"Sakanyan","year":"1993","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1007\/s11274-005-9088-y","article-title":"Purification, enzymatic properties of a recombinant D-hydantoinase and its dissociation by zinc ion","volume":"22","author":"Shi","year":"2006","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/j.tibtech.2007.03.010","article-title":"Catalytic efficiency and kcat\/Km: A useful comparator?","volume":"25","author":"Eisenthal","year":"2007","journal-title":"Trends Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1128\/jb.174.3.962-969.1992","article-title":"Cloning and sequencing of the genes involved in the conversion of 5-sustituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671","volume":"174","author":"Watabe","year":"1992","journal-title":"J. Bacteriol."},{"key":"ref_36","first-page":"92","article-title":"Characterization of thermostable hydantoinases cloned from Geobacillus stearothermophilus","volume":"113","author":"Weigel","year":"2013","journal-title":"Rep. NAS RA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1093\/genetics\/120.3.621","article-title":"Genetic applications of an inverse polymerase chain reaction","volume":"120","author":"Ochman","year":"1988","journal-title":"Genetics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"8186","DOI":"10.1093\/nar\/16.16.8186","article-title":"A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences","volume":"16","author":"Triglia","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6383","DOI":"10.1021\/bi047326v","article-title":"Structural and catalytic diversity within the amidohydrolase superfamily","volume":"44","author":"Seibert","year":"2005","journal-title":"Biochemistry"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1683","DOI":"10.1093\/nar\/gkg258","article-title":"Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties","volume":"31","author":"Goikovic","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1007\/s00775-008-0428-x","article-title":"Effect of metal binding and posttranslational lysine carboxylation on the activity of recombinant hydantoinase","volume":"14","author":"Huang","year":"2009","journal-title":"J. Biol. Inorg. Chem."},{"key":"ref_42","first-page":"9564391","article-title":"Structural Basis for pH-Dependent Oligomerization of Dihydropyrimidinase from Pseudomonas aeruginosa PAO1","volume":"2018","author":"Cheng","year":"2018","journal-title":"Bioorg. Chem. Appl."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1038\/nature08300","article-title":"Metalloproteins and metal sensing","volume":"460","author":"Waldron","year":"2009","journal-title":"Nature"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1007\/s10930-011-9343-z","article-title":"Biochemical characterization of allantoinase from Escherichia coli BL21","volume":"30","author":"Ho","year":"2011","journal-title":"Protein J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"13104","DOI":"10.1038\/s41598-018-31259-y","article-title":"A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase","volume":"8","author":"Sommer","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Schmidt, S.B., and Husted, S. (2019). The Biochemical Properties of Manganese in Plants. Plants, 8.","DOI":"10.3390\/plants8100381"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3762","DOI":"10.1074\/jbc.M513266200","article-title":"The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity","volume":"281","author":"Lohkamp","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"52","DOI":"10.3389\/fcimb.2014.00052","article-title":"Ribonucleotide reductases: Essential enzymes for bacterial life","volume":"4","author":"Torrents","year":"2014","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1300","DOI":"10.1096\/fj.06-5730rev","article-title":"DNA precursor metabolism and genomic stability","volume":"20","author":"Mathews","year":"2006","journal-title":"FASEB J."}],"container-title":["High-Throughput"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2571-5135\/9\/1\/5\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T08:57:14Z","timestamp":1760173034000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2571-5135\/9\/1\/5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,2,12]]},"references-count":49,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["ht9010005"],"URL":"https:\/\/doi.org\/10.3390\/ht9010005","relation":{},"ISSN":["2571-5135"],"issn-type":[{"value":"2571-5135","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,2,12]]}}}