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

Author

Adewale Adeboye Adewole, Stephen F Austin State UniversityFollow

Date of Award

Summer 8-8-2025

Degree Type

Thesis

Degree Name

Master of Science in Natural Science

Department

Chemistry and Biochemistry

First Advisor

Odutayo Odunuga, Ph.D.

Second Advisor

Bidisha Sengupta, Ph.D.

Third Advisor

Darrell Fry, Ph.D.

Fourth Advisor

Daniel J. Bennett, Ph.D.

Abstract

Pseudolysin (PLN) protein is a major virulent factor in Pseudomonas aeruginosa 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 Streptomyces nigrescens. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by P. aeruginosa. 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 p-nitroanilide (pNa) from N-succinyl-ala-ala-ala-p-nitroanilide (N-SucAla₃-pNa) 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 pmolpNa/sec) to hydrolyze N-SucAla₃-pNa compared to His-tagged PLN (23 pmolpNa/sec). In the presence of 1:1 stoichiometric amount of PLN to SMPI, the enzymatic activity of His-tagged PLN was enhanced to 34 pmolpNa/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 pNa/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.

Repository Citation

Adewole, Adewale Adeboye, "EFFECT OF HISTIDINE TAG ON THE ENZYMATIC ACTIVITY OF PSEUDOLYSIN AND ITS INTERACTION WITH THE STREPTOMYCES METALLOPROTEASE INHIBITOR (SMPI) PROTEIN" (2025). Electronic Theses and Dissertations. 626.
https://scholarworks.sfasu.edu/etds/626

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