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

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.

TEM and SHV Genes in Klebsiella pneumoniae Isolated from Cockroaches and Their Antimicrobial Resistance Pattern

“…Klebsiella consists of seven types— K. pneumoniae , K. planticola , K. terrigena , K. rhinoscleromatis , K. ozaenae , K. ornithinolytica , and K. oxytoca —and two of these species, K. oxytoca and K. rhinoscleromatis , have been demonstrated in human clinical specimens [10,11] . K. pneumoniae , one of the most common causes of gram-negative sepsis, usually inhabits the human and animal intestinal tract [10,20] . Some gram-negative bacilli such as K. pneumoniae and Escherichia coli strains of particular types produce β-lactamases.…”

“…Klebsiella consists of seven types, of which K. oxytoca and K. rhinoscleromatis have been demonstrated in human clinical specimens. K. pneumoniae is a gram-negative, rod-shaped, lactose-fermenting, nonmotile, and encapsulated bacillus of the Enterobacteriaceae family [10,11] . It is an opportunistic pathogen found in water, soil, and plants; it also exists as a normal flora in mucosal surfaces such as the intestines, pharynx, mouth, and skin in mammals.…”

“…It is an opportunistic pathogen found in water, soil, and plants; it also exists as a normal flora in mucosal surfaces such as the intestines, pharynx, mouth, and skin in mammals. This bacterium was also recognized as a community-acquired pulmonary pathogen, mainly among patients with a history of chronic alcoholism [10,12,13] . In humans, K. pneumoniae can colonize the gastrointestinal tract, bowel, bladder, skin, or pharynx, which may cause various clinical outcomes, including pneumonia, thrombophlebitis, bacteremia, and urinary tract infection [13] .…”

Pharmaceutical Care in a Patient with Recurrent Infection after Liver Transplant

“…K. pneumoniae [7] and Enterococcus faecium [8] were common bacteria in the human intestinal tract. Only under certain conditions could infections be induced.…”

Novel Target Exploration from Hypothetical Proteins of Klebsiella pneumoniae MGH 78578 Reveals a Protein Involved in Host-Pathogen Interaction

“…Out of the available proteome of K. pneumoniae MGH 78578, ∼11% is made up of HPs, which can be potential resources to be studied both functionally and structurally. Although bioinformatics studies on a few hypothetical proteins, such as KPN_00953(YcbK) (Teh et al, 2014), KPN_02809 (a Zinc-Dependent Metalloprotease) (Wong et al, 2012), and KPN_00728, KPN_00729 (Chain C and D of Succinate Dehydrogenase, respectively) (Choi et al, 2009), are available, mining and analysis of all hypothetical proteins to shortlist drug/vaccine targets in this pathogen is yet unexplored. In the present study, a series of in silico analyses of 540 hypothetical proteins encoded by the K. pneumoniae genome were carried out to explore novel drug/vaccine candidates in this organism.…”