{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T16:04:48Z","timestamp":1775232288767,"version":"3.50.1"},"reference-count":29,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2012,1,16]],"date-time":"2012-01-16T00:00:00Z","timestamp":1326672000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 \u00b0C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity.<\/jats:p>","DOI":"10.3390\/ijms13010901","type":"journal-article","created":{"date-parts":[[2012,1,16]],"date-time":"2012-01-16T11:14:14Z","timestamp":1326712454000},"page":"901-917","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578"],"prefix":"10.3390","volume":"13","author":[{"given":"Mun Teng","family":"Wong","sequence":"first","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sy Bing","family":"Choi","sequence":"additional","affiliation":[{"name":"Pharmaceutical Design and Simulation (PhDS) Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chee Sian","family":"Kuan","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Siang Ling","family":"Chua","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chiat Han","family":"Chang","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yahaya Mohd","family":"Normi","sequence":"additional","affiliation":[{"name":"Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei Cun","family":"See Too","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Habibah A.","family":"Wahab","sequence":"additional","affiliation":[{"name":"Pharmaceutical Design and Simulation (PhDS) Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ling Ling","family":"Few","sequence":"additional","affiliation":[{"name":"School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2012,1,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1111\/j.1365-2958.2005.04918.x","article-title":"Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model","volume":"58","author":"Lawlor","year":"2005","journal-title":"Mol. Microbiol"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1016\/j.micpath.2007.01.001","article-title":"Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia","volume":"42","author":"Lau","year":"2007","journal-title":"Microb. Pathog"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"847","DOI":"10.1016\/j.foodcont.2006.04.009","article-title":"Incidence of Klebsiella pneumonia in street foods sold in Malaysia and their characterization by antibiotic resistance, plasmid profiling, and RAPD-PCR analysis","volume":"18","author":"Haryani","year":"2007","journal-title":"Food Control"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1016\/j.jinf.2009.01.010","article-title":"Outbreak of infections due to KPC-2-producing Klebsiella pneumoniae in a hospital in Crete (Greece)","volume":"58","author":"Maltezou","year":"2009","journal-title":"J. Infect"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/0014-5793(89)80471-5","article-title":"A unique signature identifies a family of zinc-dependent metallopeptidases","volume":"242","author":"Jongeneel","year":"1989","journal-title":"FEBS Lett"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1186\/1471-2105-10-136","article-title":"Infrastructure for the life sciences: Design and implementation of the UniProt website","volume":"10","author":"Jain","year":"2009","journal-title":"BMC Bioinforma"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Liljas, A., and Pi\u0161kur, J. (2009). Textbook of Structural Biology, World Scientific.","DOI":"10.1142\/6620"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.bbagen.2005.03.016","article-title":"Cloning, expression and characterization of a 46.5-kDa metallopeptidase from Bacillus halodurans H4 sharing properties with the pitrilysin family","volume":"1725","author":"Dabonne","year":"2005","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1083\/jcb.136.2.271","article-title":"A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor","volume":"136","author":"Nouvet","year":"1997","journal-title":"J. Cell Biol"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1006\/jmbi.1998.1858","article-title":"The mitochondrial processing peptidase behaves as a zinc-metallopeptidase","volume":"280","author":"Luciano","year":"1998","journal-title":"J. Mol. Biol"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/0076-6879(95)48015-3","article-title":"Evolutionary families of metallopeptidases","volume":"248","author":"Rawlings","year":"1995","journal-title":"Methods Enzymol"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1038\/nprot.2009.2","article-title":"Protein structure prediction on the web: A case study using the Phyre server","volume":"4","author":"Kelley","year":"2009","journal-title":"Nat. Protoc"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"D211","DOI":"10.1093\/nar\/gkn785","article-title":"InterPro: The integrative protein signature database","volume":"37","author":"Hunter","year":"2009","journal-title":"Nucleic Acids Res"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1016\/j.abb.2004.08.006","article-title":"Zinc hydrolases: The mechanisms of zinc-dependent deacetylases","volume":"433","author":"Hernick","year":"2005","journal-title":"Arch. Biochem. Biophys"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4441","DOI":"10.3390\/ijms12074441","article-title":"Klebsiella pneumoniae yggG gene product: A zinc-dependent metalloprotease","volume":"12","author":"Kuan","year":"2011","journal-title":"Int. J. Mol. Sci"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Kyte, J. (2006). Structure in Protein Chemistry, Garland Science. [2nd ed].","DOI":"10.4324\/9780203833469"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"16239","DOI":"10.1021\/bi970903d","article-title":"Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme","volume":"36","author":"Bell","year":"1997","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2816","DOI":"10.1128\/JB.181.9.2816-2822.1999","article-title":"Identification of metal ligands in the Clostridium histolyticum ColH collagenase","volume":"181","author":"Jung","year":"1999","journal-title":"J. Bacteriol"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1955","DOI":"10.1002\/pro.5560041001","article-title":"Structural analysis of zinc substitutions in the active site of thermolysin","volume":"4","author":"Holland","year":"1995","journal-title":"Protein Sci"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1016\/S0014-5793(96)01412-3","article-title":"Inhibition of carboxypeptidase A by excess zinc: Analysis of the structural determinants by X-ray crystallography","volume":"400","author":"Huber","year":"1997","journal-title":"FEBS Lett"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1437S","DOI":"10.1093\/jn\/130.5.1437S","article-title":"Function and mechanism of zinc metalloenzymes","volume":"130","author":"McCall","year":"2000","journal-title":"J. Nutr"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1093\/nar\/gki198","article-title":"MAFFT version 5: Improvement in accuracy of multiple sequence alignment","volume":"33","author":"Katoh","year":"2005","journal-title":"Nucleic Acids Res"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1038\/nprot.2010.5","article-title":"I-TASSER: A unified platform for automated protein structure and function prediction","volume":"5","author":"Roy","year":"2010","journal-title":"Nat. Protoc"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"779","DOI":"10.1006\/jmbi.1993.1626","article-title":"Comparative protein modelling by satisfaction of spatial restraints","volume":"234","author":"Sali","year":"1993","journal-title":"J. Mol. Biol"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A program to check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1160","DOI":"10.1093\/bioinformatics\/btq100","article-title":"ProBiS algorithm for detection of structurally similar protein binding sites by local structural alignment","volume":"26","author":"Konc","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1002\/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B","article-title":"Automated docking using a lamarckian genetic algorithm and and empirical binding free energy function","volume":"19","author":"Morris","year":"1998","journal-title":"J. Comput. Chem"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"35289","DOI":"10.1074\/jbc.274.50.35289","article-title":"Modifying human thymidylate kinase to potentiate azidothymidine activation","volume":"274","author":"Brundiers","year":"1999","journal-title":"J. Biol. Chem"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.jmb.2006.08.084","article-title":"Elucidation of human choline kinase crystal structures in complex with the products ADP or phosphocholine","volume":"364","author":"Malito","year":"2006","journal-title":"J. Mol. Biol"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/13\/1\/901\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:48:30Z","timestamp":1760219310000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/13\/1\/901"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2012,1,16]]},"references-count":29,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2012,1]]}},"alternative-id":["ijms13010901"],"URL":"https:\/\/doi.org\/10.3390\/ijms13010901","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2012,1,16]]}}}