Biology subjects

Taylor, E. A.

Publications and source records attributed to Taylor, E. A..

6 recordsLinked to original sources

bioRxiv2026

HepI and OpsX are functionally coupled but evolutionarily asymmetric heptosyltransferase variants: ecological transitions and operon modularization drive divergent constraints and flexibility

Lipopolysaccharide (LPS) inner-core biosynthesis is classically initiated by a heptosyltransferase enzyme most commonly Heptosyltranferase I (HepI), a conserved WaaC-like enzyme. An alternative heptosyltranferase variant, OpsX, occurs alone in a subset of Gram-negative bacteria and co-exist with WaaC-like enzyme within the same genome of other organisms, raising questions about the origin of these two vairants and their functional partitioning. Here, we present a comparative evolutionary analysis of HepI (K02841) and OpsX (K12982) across Gram-negative bacteria to resolve their functional coupling and divergence. Selection analyses reveal a consistent evolutionary asymmetry, with OpsX exhibiting elevated {omega} values relative to HepI across global datasets and within genomes encoding both systems. Residue-level analyses indicate conserved catalytic cores in both enzymes, but a broader distribution of relaxed constraints in OpsX, suggesting differential partitioning of functional pressure. HepI has undergone intensified purifying selection in host-associated lineages, whereas OpsX shows no corresponding shift, indicating distinct responses to ecological context. Gene-species tree reconciliation further reveals contrasting horizontal gene transfer (HGT) architectures: HepI displays an ecologically structured network enriched in pathogen- and opportunist-associated lineages, with recurrent hub-mediated exchanges and deeper lineage-integrated events, whereas OpsX exhibits a diffuse transfer regime dominated by non-pathogenic taxa and primarily recent terminal acquisitions. These differences persist in genomes co-encoding both systems, where HepI transfer signal remains strongly associated with lifestyle, while OpsX is largely uncoupled from ecological structure. Analysis of operon architecture reveals pathway partitioning between the two genes: HepI is embedded in a conserved downstream operon linked to glycosyltransferase-mediated core assembly, whereas OpsX occurs in a more variable context enriched for upstream ADP-heptose precursor biosynthesis genes. In dual-system genomes, HepI is reduced to a minimal downstream module while OpsX retains upstream functions, indicating coordinated operon modularization. Together, HepI and OpsX form a functionally coupled but evolutionarily asymmetric system shaped by ecological transitions and genomic reorganization. HighlightsO_LIHepI and OpsX represent functionally coupled but evolutionarily asymmetric LPS inner-core biosynthesis systems across Gram-negative bacteria. C_LIO_LIOpsX shows relaxed selective constraint and a diffuse horizontal gene transfer pattern, whereas HepI is under stronger purifying selection and ecologically structured transfer. C_LIO_LIOperon organization reveals pathway modularization, with HepI embedded in conserved downstream assembly modules and OpsX retaining upstream precursor-associated flexibility. C_LI

evolutionary biology↗

bioRxiv2024

Structural Adaptations of HepI Enzymes in Proteobacteria: Insights into Evolutionary Resilience and Functional Dynamics

Since lipopolysaccharide molecules are highly variable in their structure while also being essential for bacterial cell survival, we hypothesized that enzymes involved in its biosynthesis may exhibit differences that could be used to predict the likelihood of survival in different ecological niches. We examined the sequence variability of orthologues of Heptosyltransferase I (HepI), which is found in all lipopolysaccharide (LPS) containing Gram-negative bacteria. We identified two different sequence motifs within the N-terminal domain of HepI, which correlated with differences in the Lipid A portion of the LPS. Further, we compared the protein structure of HepI from Escherichia coli with a structural model we generated that incorporated the alternate sequence motif. Molecular dynamics simulations of these two proteins, recapitulated our findings, that proteins with the E. coli-like sequence motif maintained a larger enzyme active site, while the mutated structural model undergoes rearrangements that lead to a smaller N-terminal active site pocket. This work revealed sequence-structure-function relationships that can be used to determine if a species incorporates a Heptose residue onto Lipid A chains containing one or two 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residues. Since research suggests that the number of Kdo residues in LPS impacts the overall immune response to the LPS endotoxin, this work could aid in our understanding of the pathogenic effects of human-bacterial interactions. Understanding the sequence-structural adaptations of HepI enzymes across proteobacteria sheds light on their evolutionary resilience and functional versatility. HighlightsO_LILipopolysaccharide (LPS) structure varies between organisms C_LIO_LIHeptosytransferase I sequence motifs allow prediction of LPS structural features C_LIO_LIAdaptations of HepI enzymes reveal evolutionary resilience and functional versatility C_LI

evolutionary biology↗

bioRxiv2022

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

biochemistry↗

bioRxiv2021

Discovery of Aminoglycosides as first in class, nanomolar inhibitors of Heptosyltransferase I

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis.

biochemistry↗

bioRxiv2021

Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase:Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pKa have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.

biochemistry↗

bioRxiv2020

Conserved conformational hierarchy across functionally divergent glycosyltransferases of the GT-B structural superfamily as determined from microsecond molecular dynamics

It has long been understood that some proteins to undergo conformational transitions enroute to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site is necessary for the protein to crystalize in the closed conformation. Herein we describe microsecond molecular dynamics simulations of two evolutionarily unrelated glycosytransferases, HepI and GtfA. Simulations were performed using these proteins in the open and closed conformations, (respectively,) and we sought to identify the major dynamical modes and communication networks which allow conformational transition between the open and closed structures. We provide the first reported evidence (within the scope of our experimental parameters) that conformational hierarchy/directionality of the interconversion between open and closed conformations is a conserved feature of enzymes of the same structural superfamily. Additionally, residues previously identified to be important for substrate binding in HepI were shown to have strong negative correlations with non-ionizable residues distal to the active site. Mutagenesis of these residues produced mutants with altered enzymatic efficiency exhibiting lower Km values, while the kcat is effectively unchanged. The negatively correlated motions of these residues are important for substrate binding and forming the Michaelis complex, without impacting the activation barrier for catalysis. This suggests that in the bi-domain HepI, the enzyme dynamics did not impact the transition state stabilization or chemistry, but rather earlier steps along the reaction coordinate, leading to the reorganization of the active site electrostatic environment required for catalysis.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGTglycosyltransferaseHepIheptosyltransferase IheptoseL-glycero-D-manno-heptoseADP-heptose or ADPHADP-L-glycero-D-manno-heptoseLPSlipopolysaccharide;ODLAO-deacylated E. coli Kdo2-lipid A;Trptryptophan;Proproline;Glyglycine;Argarginine;Lyslysine;CDcircular dichroism;MDMolecular Dynamics;PDBProtein Data BankPCAPrincipal Component Analysis,DCCDynamic Cross CorrelationView Full Text

biophysics↗