About

Margaret Glasner is an Associate Professor in the Department of Biochemistry and Biophysics at Texas A&M University. Her research focuses on the principles of protein evolution, enzyme promiscuity, and metabolic pathway evolution. Her laboratory employs molecular evolution, bioinformatics, and protein chemistry to investigate the evolution and biophysical basis of enzyme specificity, aiming to understand the relationship between protein structure and function from atomic to genomic levels. A major aspect of her work involves studying catalytic promiscuity—the ability of enzymes to catalyze different chemical reactions using the same active site—and how this property serves as raw material for evolving new enzyme activities. Her research explores how promiscuous enzymes can be recruited to develop new metabolic pathways, with particular attention to the mechanistic basis of enzyme promiscuity, including substrate orientation and reactivity of catalytic amino acids. Glasner's work also investigates the biophysical basis of intramolecular epistasis, examining how mutations affect enzyme stability, folding, and activity in different sequence contexts. She uses ancestral sequence reconstruction, structural comparisons, and experimental methods to map epistatic interactions within enzyme families. Additionally, her research extends to the evolution of new metabolic pathways through underground metabolism, where she studies the role of promiscuous enzyme activities and horizontal gene transfer in metabolic innovation. Her contributions aim to identify fundamental evolutionary principles that can inform protein structure-function relationships, protein and metabolic engineering, and the understanding of enzyme evolution.

Research topics

• Chemistry
• Biochemistry
• Biology

Selected publications

• Intramolecular epistasis correlates with divergence of specificity in promiscuous and bifunctional <scp>NSAR</scp>/<scp>OSBS</scp> enzymes

  Protein Science · 2025-04-18 · 1 citations

  articleOpen accessSenior author

  Understanding the functions and evolution of specificity-determining residues is essential for improving strategies to predict and design enzyme functions. Whether the function of an amino acid residue is retained during evolution depends on intramolecular epistasis, which occurs when the same residue contributes to different phenotypes in different genetic backgrounds. This study examines the relationship between epistasis and functional divergence by investigating a conserved specificity determinant in five homologs from the N-succinylamino acid racemase (NSAR)/o-succinylbenzoate synthase (OSBS) subfamily. NSAR activity originated as a promiscuous (non-biological) activity of an ancestral OSBS. Some extant NSAR/OSBS subfamily enzymes still have OSBS activity as a biological function and NSAR as a promiscuous activity, while some use both OSBS and NSAR activities as biological functions. Others use only NSAR activity as a biological function but can still catalyze the OSBS reaction as a promiscuous activity. Previously, we determined that the conserved residue R266 in Amycolatopsis sp. T-1-60 NSAR contributes to NSAR specificity by enabling K263 to act as a general acid/base catalyst. Here, we show that mutating R266 decreased relative specificity for NSAR activity in four of five NSAR/OSBS subfamily enzymes, as predicted. However, other phenotypes exhibited epistasis related to the pleiotropy of R266, including the proton exchange rate between the catalytic lysines and the substrate, the impact on OSBS activity, and thermostability. The strength of epistasis was associated with functional and evolutionary divergence of NSAR/OSBS enzymes. These results illustrate the benefits of comparing multiple homologs for understanding mechanisms of enzyme specificity.

  PublisherOA PDFDOI

• Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily

  bioRxiv (Cold Spring Harbor Laboratory) · 2022

  Senior authorCorresponding
  • Chemistry

  Abstract Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N -succinylamino acid racemase/ o -succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al ., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.

  PublisherOA PDFDOI

• Second-Shell Amino Acid R266 Helps Determine <i>N</i>-Succinylamino Acid Racemase Reaction Specificity in Promiscuous <i>N</i>-Succinylamino Acid Racemase/<i>o</i>-Succinylbenzoate Synthase Enzymes

  Biochemistry · 2021 · 5 citations

  Senior authorCorresponding
  • Chemistry
  • Biochemistry
  • Biology

  NSAR/OSBS profoundly reduces NSAR activity but moderately reduces OSBS activity. This is due to a 1000-fold decrease in the rate of proton exchange between the substrate and the general acid/base catalyst K263. This mutation is less deleterious for the OSBS reaction because K263 forms a cation-π interaction with the OSBS substrate and/or the intermediate, rather than acting as a general acid/base catalyst. Together, the data explain how R266 contributes to NSAR reaction specificity and was likely an essential preadaptation for the evolution of NSAR activity.

  PublisherOA PDFDOI

• Oxidative opening of the aromatic ring: Tracing the natural history of a large superfamily of dioxygenase domains and their relatives

  Journal of Biological Chemistry · 2019-05-16 · 25 citations

  articleOpen access

  -adenosylmethionine family domain. These observations point to two distinct yet potentially overlapping contexts wherein the elusive molecular function of the Memo domain could be finally resolved, thereby linking it to nucleotide base and aliphatic isoprenoid modification. In total, this report throws light on the functions of large swaths of the experimentally-uncharacterized PCAD-Memo families.

  PublisherOA PDFDOI

• How enzyme promiscuity and horizontal gene transfer contribute to metabolic innovation

  FEBS Journal · 2019-12-20 · 75 citations

  reviewOpen access1st authorCorresponding

  Promiscuity is the coincidental ability of an enzyme to catalyze its native reaction and additional reactions that are not biological functions in the same active site. Promiscuity plays a central role in enzyme evolution and is thus a useful property for protein and metabolic engineering. This review examines enzyme evolution holistically, beginning with evaluating biochemical support for four enzyme evolution models. As expected, there is strong biochemical support for the subfunctionalization and innovation-amplification-divergence models, in which promiscuity is a central feature. In many cases, however, enzyme evolution is more complex than the models indicate, suggesting much is yet to be learned about selective pressures on enzyme function. A complete understanding of enzyme evolution must also explain the ability of metabolic networks to integrate new enzyme activities. Hidden within metabolic networks are underground metabolic pathways constructed from promiscuous activities. We discuss efforts to determine the diversity and pervasiveness of underground metabolism. Remarkably, several studies have discovered that some metabolic defects can be repaired via multiple underground routes. In prokaryotes, metabolic innovation is driven by connecting enzymes acquired by horizontal gene transfer (HGT) into the metabolic network. Thus, we end the review by discussing how the combination of promiscuity and HGT contribute to evolution of metabolism in prokaryotes. Future studies investigating the contribution of promiscuity to enzyme and metabolic evolution will need to integrate deeper probes into the influence of evolution on protein biophysics, enzymology, and metabolism with more complex and realistic evolutionary models. ENZYMES: lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), OSBS (EC 4.2.1.113), HisA (EC 5.3.1.16), TrpF, PriA (EC 5.3.1.24), R-mandelonitrile lyase (EC 4.1.2.10), Maleylacetate reductase (EC 1.3.1.32).

  PublisherOA PDFDOI

• Comparison of <i>Alicyclobacillus acidocaldarius</i> <i>o</i>-Succinylbenzoate Synthase to Its Promiscuous <i>N</i>-Succinylamino Acid Racemase/<i>o</i>-Succinylbenzoate Synthase Relatives

  Biochemistry · 2018-05-16 · 10 citations

  articleOpen accessSenior authorCorresponding

  Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M–1 s–1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

  PublisherOA PDFDOI

• Finding enzymes in the gut metagenome

  Science · 2017-02-10 · 15 citations

  letter1st authorCorresponding

  Metagenomic data help to elucidate enzymatic pathways in the human gut microbiome

  PublisherDOI

• Role of an Active Site Loop in the Promiscuous Activities of <i>Amycolatopsis</i> sp. T-1-60 NSAR/OSBS

  Biochemistry · 2014-06-23 · 12 citations

  articleSenior author

  The o-succinylbenzoate synthase (OSBS) family is part of the functionally diverse enolase superfamily. Many proteins in one branch of the OSBS family catalyze both OSBS and N-succinylamino acid racemization in the same active site. In some promiscuous NSAR/OSBS enzymes, NSAR activity is biologically significant in addition to or instead of OSBS activity. Identifying important residues for each reaction could provide insight into how proteins evolve new functions. We have made a series of mutations in Amycolatopsis sp. T-1-60 NSAR/OSBS in an active site loop, referred to as the 20s loop. This loop affects substrate specificity in many members of the enolase superfamily but is poorly conserved within the OSBS family. Deletion of this loop decreased OSBS and NSAR catalytic efficiency by 4500-fold and 25,000-fold, respectively, showing that it is essential. Most point mutations had small effects, changing the efficiency of both NSAR and OSBS activities <10-fold compared to that of the wild type. An exception was F19A, which reduced kcat/KM(OSBS) 200-fold and kcat/KM(NSAR) 120-fold. Mutating the surface residue R20E, which can form a salt bridge to help close the 20s loop over the active site, had a more modest effect, decreasing kcat/KM of OSBS and NSAR reactions 32- and 8-fold, respectively. Several mutations increased KM of the NSAR reaction more than that of the OSBS reaction. Thus, both activities require the 20s loop, but differences in how mutations affect OSBS and NSAR activities suggest that some substitutions in this loop made a small contribution to the evolution of NSAR activity, although additional mutations were probably required.

  PublisherDOI

• Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

  Proceedings of the National Academy of Sciences · 2014-05-28 · 30 citations

  articleOpen accessSenior author

  The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.

  PublisherOA PDFDOI

• Promiscuity of Exiguobacterium sp. AT1b o-succinylbenzoate synthase illustrates evolutionary transitions in the OSBS family

  Biochemical and Biophysical Research Communications · 2014-06-16 · 11 citations

  articleSenior authorCorresponding
  PublisherDOI

Recent grants

• NIH Grant P41RR001081

  NIH · $4.6M · 2012

• Biophysical constraints on evolution of enzyme specificity

  NIH · $1.2M · 2018–2024

• CAREER: Evolutionary Mechanisms for Recruiting Promiscuous Enzyme Activities into New Metabolic Pathways

  NSF · $716k · 2013–2019

Frequent coauthors

• David P. Bartel

  Whitehead Institute for Biomedical Research

  47 shared

• Ryan M. Cinalli

  Icahn School of Medicine at Mount Sinai

  36 shared

• Scott Baskerville

  36 shared

• Antonio J. Giráldez

  Yale University

  36 shared

• Scott M. Hammond

  University of North Carolina at Chapel Hill

  36 shared

• Anton J. Enright

  University of Cambridge

  36 shared

• J. Michael Thomson

  University of Edinburgh

  36 shared

• Alexander F. Schier

  University of Basel

  36 shared

Labs

• Department of Biochemistry and BiophysicsPI

Education

• B.S.

  University of Wyoming

  1995

• Other

  University of Wyoming

  1995

• Ph.D.

  Massachusetts Institute of Technology

  2003

• Other

  University of California, San Francisco

  2008