4DEL

Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from M. tuberculosis

Summary for 4DEL

• Entry DOI: 10.2210/pdb4del/pdb
• Related: 3EKL 3EKZ 3ELF 4DEF
• Descriptor: Fructose-bisphosphate aldolase, SODIUM ION, ZINC ION, ... (7 entities in total)
• Functional Keywords: class ii fructose-1, 6-bisphosphate aldolase, zinc enzyme, dihydroxyacetone, glyceraldehyde-3-phosphate, aldol condensation, glycolysis, lyase, metal-binding, phosphoglycolohydroxamate
• Biological source: Mycobacterium tuberculosis
• Total number of polymer chains: 1
• Total formula weight: 37942.33

Authors

Pegan, S.D.,Mesecar, A.D. (deposition date: 2012-01-20, release date: 2013-01-23, Last modification date: 2023-09-13)

Primary citation

Pegan, S.D.,Rukseree, K.,Capodagli, G.C.,Baker, E.A.,Krasnykh, O.,Franzblau, S.G.,Mesecar, A.D.
Active site loop dynamics of a class IIa fructose 1,6-bisphosphate aldolase from Mycobacterium tuberculosis.
Biochemistry, 52:912-925, 2013

PubMed Abstract: Class II fructose 1,6-bisphosphate aldolases (FBAs, EC 4.1.2.13) comprise one of two families of aldolases. Instead of forming a Schiff base intermediate using an ε-amino group of a lysine side chain, class II FBAs utilize Zn(II) to stabilize a proposed hydroxyenolate intermediate (HEI) in the reversible cleavage of fructose 1,6-bisphosphate, forming glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). As class II FBAs have been shown to be essential in pathogenic bacteria, focus has been placed on these enzymes as potential antibacterial targets. Although structural studies of class II FBAs from Mycobacterium tuberculosis (MtFBA), other bacteria, and protozoa have been reported, the structure of the active site loop responsible for catalyzing the protonation-deprotonation steps of the reaction for class II FBAs has not yet been observed. We therefore utilized the potent class II FBA inhibitor phosphoglycolohydroxamate (PGH) as a mimic of the HEI- and DHAP-bound form of the enzyme and determined the X-ray structure of the MtFBA-PGH complex to 1.58 Å. Remarkably, we are able to observe well-defined electron density for the previously elusive active site loop of MtFBA trapped in a catalytically competent orientation. Utilization of this structural information and site-directed mutagenesis and kinetic studies conducted on a series of residues within the active site loop revealed that E169 facilitates a water-mediated deprotonation-protonation step of the MtFBA reaction mechanism. Also, solvent isotope effects on MtFBA and catalytically relevant mutants were used to probe the effect of loop flexibility on catalytic efficiency. Additionally, we also reveal the structure of MtFBA in its holoenzyme form.

PubMed: 23298222
DOI: 10.1021/bi300928u

Experimental method

X-RAY DIFFRACTION (1.58 Å)