EFFECT OF HISTIDINE TAG ON THE ENZYMATIC ACTIVITY OF PSEUDOLYSIN AND ITS INTERACTION WITH THE STREPTOMYCES METALLOPROTEASE INHIBITOR (SMPI) PROTEIN

<p>Pseudolysin (PLN) protein is a major virulent factor in <em>Pseudomonas</em> <em>aeruginosa </em>infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by <em>Streptomyces nigrescens</em>. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by <em>P</em>. <em>aeruginosa</em>. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography and gel filtration. His tag was removed by digestion with tobacco etch virus (TEV) protease. Proteolytic activity of tagged or untagged PLN, monitored as the rate of release of <em>p</em>-nitroanilide (<em>p</em>Na) from <em>N</em>-succinyl-ala-ala-ala-<em>p</em>-nitroanilide (<em>N</em>-SucAla₃-<em>p</em>Na) at 410 nm, was assayed in the presence and absence of SMPI. Analysis of data showed that in the absence of SMPI, untagged PLN was two-fold more active (46 pmol<em>p</em>Na/sec) to hydrolyze <em>N</em>-SucAla₃-<em>p</em>Na compared to His-tagged PLN (23 pmol<em>p</em>Na/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmol<em>p</em>Na/sec while that of untagged PLN remained the same. Doubling the concentration of SMPI (ratio 1:2 PLN to SMPI) reduced the enzymatic activity of untagged PLN by 50 % (23 pmol <em>p</em>Na/sec). The outcomes of this study implied that His tag at the N terminus of PLN impacted its enzymatic activity and its interaction with SMPI.</p>

Name thesis:degree_name

Master of Science in Natural Science

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Chemistry and Biochemistry

Year dc:date.available

2025

Author dc:creator

• Adewole, Adewale Adeboye

Contributors dc:contributor

• Odutayo Odunuga, Ph.D.
• Bidisha Sengupta, Ph.D.
• Darrell Fry, Ph.D.

• Pseudolysin
• Drug Development
• Pseduomonas aeruginosa
• Streptomyces metalloprotease inhibitors
• antimicrobial resistance
• Protein interactions
• enzymatic assay
• Biochemistry
• Molecular Biology
• Pathogenic Microbiology
• Pharmaceutical Preparations
• Pharmacology

Repository record dc:identifier

https://scholarworks.sfasu.edu/etds/626

OAI identifier oai:identifier

oai:scholarworks.sfasu.edu:etds-1688