Structural variations of transketolases over the evolution from bacteria to higher eukaryotes: In silico/in vitro...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Transketolases (TKs) are crucial enzymes in the pentose phosphate pathway, vital for synthesizing amino acids, nucleotides, and managing oxidative stress.
• Targeting pathogen-specific TKs offers a potential route for novel antibiotic development by exploiting differences between human and microbial enzymes.

Purpose of the Study:

• To compare the structural characteristics of human transketolase (hTK) with those of thirteen transketolases from priority pathogenic organisms.
• To identify potential structural differences that could be exploited for the development of selective TK inhibitors as antibiotics.

Main Methods:

• Expression and purification of human and thirteen pathogenic transketolases.
• X-ray diffraction to determine six new experimental structures.
• In silico modeling for electron density map resolution and validation.
• Comparative analysis of active sites and monomer-monomer interfaces.

Main Results:

• Structural comparison revealed that animal TKs are significantly shorter (at least 50 residues) and have evolved differently from bacterial, fungal, and parasitic TKs.
• The monomer-monomer interface is highly specific across different transketolases, whereas the active site is more conserved.
• Human transketolase exhibits a higher number of non-covalent bonds in both active sites and interfaces compared to pathogen transketolases, likely to stabilize its shorter structure.

Conclusions:

• Pathogen transketolases possess distinct structural features, particularly at the monomer-monomer interface, compared to human transketolase.
• These structural differences suggest that pathogen transketolases can be selectively inhibited, potentially via targeting the monomer-monomer interface, without impacting human transketolase activity.
• This research provides a structural basis for designing novel antibiotics that specifically target microbial transketolases.