Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

Abstract

Enzymes are known to adopt various conformations at different points along their catalytic cycles. Here, we present a comprehensive analysis of 15 isomorphous, high resolution crystal structures of the enzyme phosphoglucomutase from the bacterium Xanthomonas citri . The protein was captured in distinct states critical to function, including enzyme-substrate, enzyme-product, and enzyme-intermediate complexes. Key residues in ligand recognition and regions undergoing conformational change are identified and correlated with the various steps of the catalytic reaction. In addition, we use principal component analysis to examine various subsets of these structures with two goals: (1) identifying sites of conformational heterogeneity through a comparison of room temperature and cryogenic structures of the apo-enzyme and (2) a priori clustering of the enzyme-ligand complexes into functionally related groups, showing sensitivity of this method to structural features difficult to detect by traditional methods. This study captures, in a single system, the structural basis of diverse substrate recognition, the subtle impact of covalent modification, and the role of ligand-induced conformational change in this representative enzyme of the α-D-phosphohexomutase superfamily.

Structural basis for substrate and product recognition in human phosphoglucomutase-1 (PGM1) isoform 2, a member of the α-d-phosphohexomutase superfamily

“…It is less clear from available structures (present results and in Stiers et al 24 and references cited therein) why the PMM/PGMs have roughly the same activity on both α-d-mannose and α-d-glucose 1-phosphate substrates, while the PGMs have an activity that is several orders of magnitude higher for the latter 7,32 . Firstly, in the X. citri PMM/PGM:G1P complex there is an Arg residue interacting with hydroxyls both at C2 and C3 24 , but this residue is not conserved in metazoan PGMs. It is therefore not a universal axial C2-hydroxyl "reader", and in our human PGM1-2:G1P complex there are no residues interacting directly with the C2 hydroxyl.…”

“…Only minor differences can be seen when comparing the free protein structure with the complex structures, with D4 rotated 2° or www.nature.com/scientificreports www.nature.com/scientificreports/ less in the complexes, compared to the free protein structure. Similarly, in a recent study of complex structures and free protein for a bacterial phosphoglucomutase 24 conformational variability was not observed for D4. It was speculated that this might be due to tight packing in the crystal lattice.…”

“…The only part of the hexose substrate that is not tightly and specifically interacting with either the phosphate-binding, sugar-binding, or p-Ser containing loops is the C2 carbon and its substituent, explaining that only at this position is there variation in substrate specificity in the PHM superfamily. The Beamer group recently published 15 crystal structures of a PMM/PGM from the γ-proteobacterium Xanthomonas citri, including complexes with 1-and 6-phosphohexoses and the G16P intermediate 24 . This work confirms that the loops and residues involved in PHM catalysis are conserved from bacteria to human.…”

Crystal structures of Schistosoma mansoni histone deacetylase 8 reveal a novel binding site for allosteric inhibitors

“…PCA has been proved to detect conformational differences in protein structures, which ultimately correlate to functional activities ( 31 ). PCA was recently applied to get insights into the conformational ensembles and space sampling of GroEL ( 32 ), heterotrimeric G proteins ( 33 ), and Xanthomonas citri phosphohexomutase structures ( 34 ). Hence, PCA was performed on the best-fit structural alignment of all our structures; as a result, the first three PCs accounted for 98% of the total variance ( Fig.…”

Drug Design: Where We Are and Future Prospects

“…Working on a single crystal is marginally useful to understanding the enzyme movements during the catalytic process and to plan possible molecular structures interacting or interfering with different conformational states of the enzyme. At the moment, it is possible to combine different crystal snapshots to have an idea of the enzyme conformational changes during the catalytic process, as it was performed for the ubiquitous enzymes α-D-phosphohexomutases [23].…”