Structural and biochemical studies on salmonella typhimurium aldose 1-epimerase

Abstract

Mutarotase plays a key role in carbohydrate metabolism by catalyzing the reversible conversion of - and -anomers of aldose sugars. In this thesis, I report the X-ray crystallographic analysis of Salmonella typhimurium aldose 1-epimerase (sfYeaD) in two different crystal forms. The work is presented in four chapters, each focusing on different aspects of the study.

Chapter 1 - Introduction Carbohydrates exhibit remarkable functional diversity due to their structural complexity, chirality, and the variety of linkages they can form. This chapter highlights how these features contribute to the vast biological roles of carbohydrates.

Chapter 2 - Protein Expression and Purification sfYeaD with an N-terminal hexahistidine tag was overexpressed in E. coli BL21 (DE3) pLysS.

Purification was achieved in a single step using Ni-NTA affinity chromatography.

Preliminary biochemical and biophysical analyses (mass spectrometry, gel filtration, dynamic light scattering) confirmed suitability for crystallization.

Chapter 3 - Crystallographic Analysis sfYeaD crystallized using the hanging drop vapor diffusion method at 20 °C in three different crystal forms.

Crystals diffracted to ~2.5 Å resolution (C-centered monoclinic form) and 2.9 Å resolution (primitive orthorhombic form).

Structure solved using molecular replacement (MR).

The polypeptide chain folds into an intricate array of 22 -strands (3 -sheets) and 5 small -helices.

Chapter 4 - Comparative Structural Studies Comparison of sfYeaD complex structures with those of GalM (galactose mutarotase) bound to different ligands.

Essential catalytic residues were identified, revealing both structural and mechanistic similarities between sfYeaD and GalM.

Despite similarities, major differences in substrate-binding residues were observed, likely determining the specificity of the YeaD class.

Conclusion This study provides new insights into the structural basis of mutarotase activity in Salmonella typhimurium. The identification of conserved and variable residues highlights both evolutionary conservation and functional divergence within the mutarotase family.