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

Mun Teng Wong,Wei Cun See Too,Habibah A Wahab,Ling Ling Few,Chee Sian Kuan,Yahaya Mohd Normi,Chiat Han Chang,Sy Bing Choi,Siang Ling Chua

doi:10.3390/ijms13010901

Mun Teng Wong, Wei Cun See Too + Show 7 more

Open Access

https://doi.org/10.3390/ijms13010901

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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.

Highlights

IntroductionKlebsiella was first identified as a cause of pneumonia in 1882 by a pathologist Karl Friedlander [1]
Klebsiella was first identified as a cause of pneumonia in 1882 by a pathologist Karl Friedlander [1].Klebsiella pneumoniae is a Gram negative; rod shaped and encapsulated bacterium of the familyEnterobacteriaceae, which normally inhabits the animal and human intestinal tract [2]
In this work, besides presenting results from computational approaches to model the structure of this hypothetical protein in order to elucidate its function, we report the cloning and expression of the open reading frame of ypfJ gene that encodes for this hypothetical protein

Summary

Introduction

Klebsiella was first identified as a cause of pneumonia in 1882 by a pathologist Karl Friedlander [1]. K. pneumoniae strain MGH 78578 is one of the strains that show high level of resistance to multiple antimicrobial agents including ampicillin, oxacillin, kanamycin, and chloramphenicol [5] This strain was originally isolated from the sputum of a male patient in 1994 [5] and its genome has been sequenced by the Genome Sequencing Center of Washington University in Saint Louis in 2007. Zinc metalloproteases catalyze peptide bond hydrolysis in a protein or peptide substrate They contain divalent metal ions on their active sites; activate the water molecule as the direct attacking species on the peptide bond. Despite its predicted function as a metalloprotease, the protease activity of KPN_02809 has never been experimentally confirmed and it is still being designated as a hypothetical metalloprotease This ypfJ gene product has never been investigated experimentally. Characterization of the purified recombinant protein supported the results of the bioinformatics approaches

Results and Discussion

Molecular Docking Simulation

Experimental Section

Reverse Transcription PCR of ypfJ Gene

Casein Hydrolysis Assay

Conclusions