Analysis of the Structural Determinants Underlying Discrimination between Substrate and Solvent in β-Phosphoglucomutase Catalysis

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Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

Section: Resultsmentioning

confidence: 59%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

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124

139

“…In structural terms, the open conformation of the GSA complex is suitable for access of the substrate to the active site, whereas the general base that promotes reactivity (the D10 side chain) is orientated away from its position in the TSA complex. Such disruption of general base catalysis should reduce the rate of hydrolysis of the phosphoenzyme resulting from the inadvertent activation of water molecules (19,20,28,29). Indeed, the high resolution of the PGM-BeF 3 GSA complex structure (1.65 Å) allows the arrangement of water molecules in the active site to be determined accurately.…”

Section: Discussionmentioning

confidence: 99%

Near attack conformers dominate β-phosphoglucomutase complexes where geometry and charge distribution reflect those of substrate

Griffin

Bowler

Baxter

et al. 2012

Experimental observations of fluoromagnesate and fluoroaluminate complexes of β-phosphoglucomutase (β-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of β-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of β-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.ground-state analogues | enzyme mechanism T he stabilization of transition states (TS) is central to the high reaction rates achieved by enzymes (1-3). Most studies to date have focused on TSs that include a chemical transformation step, where the reacting species have some partial chemical bonding character. However, enzymes also have to avoid substantial barriers associated with nonchemical transformation steps and hence may be required to stabilize other regions of the complex energy surface corresponding to the reaction coordinate. The concept of near attack conformers (NACs) was introduced as a useful tool to help analyze these different roles, by partitioning chemical transformation and nonchemical transformation steps (4). NACs were defined as species where the reactants have an appropriate geometry for the development of the chemical TS (5, 6). Furthermore, the component of the TS barrier ascribed to the chemical TS was proposed to be relatively constant within systems carrying out similar chemistry, on the basis of a linear relationship between the (calculated) population of NACs and the corresponding reaction rates (5). Hence, in free-energy terms, the NAC model apportions the TS barrier (ΔG † ) between a variable conformational component (ΔG NAC ) and an invariant chemical (covalent) component (ΔG chem ; Fig. 1). For some uncatalyzed reactions, it was proposed that a major component of the TS free-energy barrier is the population of NACs, t...

“…mechanical -molecular mechanical (QM-MM) analysis based on the structure (19) and was subsequently validated in solution by direct observation based on 19 F NMR data (8). However, the MgF − 3 interpretation was questioned (20,21) and the pentacovalent phosphorane interpretation defended (22,23), despite the presence of fluoride, which severely compromises the catalytic cycle of β-PGM (15). Therefore, using an integration of NMR and high-resolution x-ray structural data, we now report a thorough characterization of β-PGM in the presence of G6P and fluoride that leaves no room for a pentacovalent phosphorane interpretation of solid-state or solution-state complexes.…”

mentioning

confidence: 99%

Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF3- rather than by phosphoranes

Baxter

Bowler

Alizadeh

et al. 2010

137

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO − 3 , but is MgF − 3 (trifluoromagnesate). Although MgF − 3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19 F NMR. Measurements on three β-PGM-MgF − 3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F⋯H-N scalar couplings, and the atomic positions determined from the highresolution crystal structure of the β-PGM-MgF − 3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF − 3 model of near-transition state conformations.19F NMR | phosphoryl transfer enzyme | transition state analogue | trifluoromagnesate T he mono-and diesters of phosphoric acid have commanding and ubiquitous roles in all species of life. As structural components they show remarkable stability to spontaneous hydrolysis under near physiological conditions (25°C), with half-lives for P-O bond cleavage in phosphate diesters estimated at ca. 10 7 years and for monoesters ca. 10 12 years (1, 2). Yet, they are susceptible to enzyme-catalyzed hydrolysis and phosphoryl group transfer reactions either between two oxygens, or between oxygen and nitrogen or sulfur, with turnover numbers adequate to support a vast array of biological processes, e.g. Serratia nuclease k cat ca. 2; 500 s −1 (3), E. coli alkaline phosphatase k cat ≥ 45 s −1 (4), and human protein tyrosine phosphatase β k cat ca.