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Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis
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Document Title
Active Site Loop Dynamics of a Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis
Author
Pegan SD, Rukseree K, Capodagli GC, Baker EA, Krasnykh O, Franzblau SG, Mesecar AD
Name from Authors Collection
Affiliations
University of Denver; University of Denver; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC); University of Illinois System; University of Illinois Chicago; University of Illinois Chicago Hospital; University of Illinois System; University of Illinois Chicago; University of Illinois Chicago Hospital; Purdue University System; Purdue University; Purdue University West Lafayette Campus; Purdue University System; Purdue University; Purdue University West Lafayette Campus; Purdue University System; Purdue University; Purdue University West Lafayette Campus
Type
Article
Source Title
BIOCHEMISTRY
Year
2013
Volume
52
Issue
5
Page
912-925
Open Access
Green Accepted
Publisher
AMER CHEMICAL SOC
DOI
10.1021/bi300928u
Format
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 epsilon-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 angstrom. 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.
Industrial Classification
Knowledge Taxonomy Level 1
Knowledge Taxonomy Level 2
Knowledge Taxonomy Level 3
Funding Sponsor
National Center for Genetic Engineering and Biotechnology, Thailand; Professional Research Opportunities for Faculty; Colorado Center for Drug Discovery; Walther Cancer Foundation; Southeast Regional Collaborative Access Team (SER-CAT) [22-ID]; U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-06CH11357, W-31-109-Eng-38]; Michigan Economic Development Corp.; Michigan Technology Tr-Corridor [085P1000817]; NATIONAL CANCER INSTITUTE [P30CA023168] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R03AI092249] Funding Source: NIH RePORTER
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