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Int. J. Mol. Sci. 2012, 13(1), 901-917; doi:10.3390/ijms13010901
Article

Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578

1
, 2
, 1
, 1
, 1
, 3
, 1
, 2,*  and 1,*
1 School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian 16150, Kelantan, Malaysia 2 Pharmaceutical Design and Simulation (PhDS) Laboratory, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden 11800, Pulau Pinang, Malaysia 3 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia
* Authors to whom correspondence should be addressed.
Received: 27 October 2011 / Revised: 29 December 2011 / Accepted: 9 January 2012 / Published: 16 January 2012
(This article belongs to the Special Issue Hypothetical Proteins)
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Abstract

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 °C 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.
Keywords: Klebsiella pneumoniae MGH 78578; hypothetical protein; homology modeling; molecular docking simulation; metalloprotease; metalloprotease inhibitors; ypfJ gene Klebsiella pneumoniae MGH 78578; hypothetical protein; homology modeling; molecular docking simulation; metalloprotease; metalloprotease inhibitors; ypfJ gene
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Wong, M.T.; Choi, S.B.; Kuan, C.S.; Chua, S.L.; Chang, C.H.; Normi, Y.M.; See Too, W.C.; Wahab, H.A.; Few, L.L. Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578. Int. J. Mol. Sci. 2012, 13, 901-917.

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