Margaret Glasner

Associate Professor | Biochemistry & Biophysics

protein evolution, enzyme promiscuity, metabolic pathway evolution, metabolic pathway discovery, comparative genomics

Office:BICH / 237A

Evolution is the organizing principle of biology and provides the cornerstone of our approach to understand the relationships between protein structure and function. We combine bioinformatics, biochemistry, and genetics to address fundamental questions about protein evolution. Our primary focus is to discover how catalytic promiscuity serves as the raw material for evolving new enzyme activities. Catalytic promiscuity is the ability to catalyze different chemical reactions using the same active site. We study this problem on two fronts: understanding the evolutionary potential of catalytically promiscuous proteins and discovering how enzymes that have promiscuous activities can be recruited to evolve new metabolic pathways. Our goal is to use results from our research to identify fundamental evolutionary principles that can help decipher protein structure-function relationships, predict protein functions, and improve protein or metabolic engineering methods.

Mechanistic basis of catalytic promiscuity

Identification of a specificity determinant in an NSAR/OSBS enzyme. A) The OSBS family can be divided into several structurally distinct subfamilies. NSAR activity evolved in one subfamily, shown in red. B) The OSBS and NSAR reactions use structurally similar substrates to catalyze different different reactions. C) Sequence logos of each subfamily show that R266 is conserved in the NSAR/OSBS subfamily, while position 266 is primarily nonpolar in other OSBS subfamilies. D) Hydrogen-deuterium exchange experiments show that mutating the conserved R2666 (top) to glutamine (bottom) severely reduces the rate of proton exchange between the catalytic K263 and the substrate.
See Biochemistry 51, 6171–6181 (2012).

We are investigating the mechanistic basis of promiscuity in enzymes that catalyze O-succinylbenzoate synthesis (OSBS) and N-succinylamino acid racemization (NSAR). NSAR activity originated as a promiscuous activity in one subfamily of the large, diverse OSBS enzyme family. Our results so far indicate that substrate orientation and reactivity of catalytic amino acids, rather than substrate-binding affinity, are the keys to catalytic promiscuity of NSAR/OSBS enzymes. Ancestral sequence reconstruction and structural comparisons identified a tyrosine to isoleucine/leucine mutation, which modified the shape and size of the active site to enable N-succinylamino acids to bind in the right orientation for catalysis. Comparing sequence conservation in the NSAR/OSBS subfamily to other OSBS subfamilies, which lack NSAR activity, identified a second-shell arginine that is also essential for NSAR activity in Amycolatopsis sp. T-1-60 NSAR/OSBS. Mutating this arginine showed that it determines the reactivity of an adjacent catalytic lysine, a general acid-base catalyst in the NSAR reaction, but serves as a cation to stabilize the transition state in the OSBS reaction. We are continuing to search for and evaluate other mutations necessary for the evolution of NSAR activity using ancestral reconstruction, sequence/structure comparisons, and library screening approaches. 

D. Odokonyero, A. W. McMillan, U. A. Ramagopal, R. Toro, D. P. Truong, M. Zhu, M. S. Lopez, B. Somiari, M. Herman, A. Aziz, J. B. Bonanno, K. G. Hull, S. K. Burley, D. Romo, S. C. Almo, M. E. Glasner, Comparison of alicyclobacillus acidocaldarius O-succinylbenzoate synthase to its promiscuous N-succinylamino acid racemase/ O-succinylbenzoate synthase relatives. Biochemistry 57, 3676–3689 (2018).

Biophysical basis of intramolecular epistasis

Intramolecular epistasis occurs when mutations have different effects in different sequence contexts; as a result, mutations within a protein have non-additive effects. Recent studies demonstrate that epistasis has a major influence on evolutionary trajectories of proteins. As a result, identifying epistatic interactions and understanding how they affect protein structure-function relationships is essential for understanding how proteins carry out their functions and engineering proteins. Catalytically promiscuous enzymes offer an unprecedented opportunity to investigate the biophysical basis of epistasis because epistatic mutations that affect stability and folding are likely to affect all the enzyme’s activities. In contrast, epistatic mutations that affect catalysis may alter the relative specificity among the activities. For example, in contrast to the preferential effect of R266Q on NSAR activity in Amycolatopsis sp. T-1-60 NSAR/OSBS, R266Q mutations in several other enzymes are highly deleterious for both NSAR and OSBS activities, indicating a broader role in protein conformation or stability. We are using computational and experimental methods to broadly map and characterize the effects of epistatic interactions within NSAR/OSBS enzymes. This will provide a comprehensive understanding of how epistatic interactions among residues influence protein structure-function relationships.

Underground metabolism in the evolution of new metabolic pathways

Comparative genomics shows that NSAR genes occur in a variety of operon contexts. A) Characterized pathway that utilizes NSAR activity. B) Genome context network showing variations among the genes that are encoded next to an NSAR gene.
See Biochemistry 45, 4455–4462.

Metabolic pathways require the coordinated expression, substrate specificity, and kinetic properties of multiple enzymes. As a result, the evolution of new metabolic pathways is a complicated problem, requiring several low-probability events. Catalytic promiscuity and broad substrate specificity comprise an “underground metabolism” of side reactions that could be recruited to form new metabolic pathways. Events, such as mutations that alter flux through an underground reaction or horizontal gene transfer, could solidify connections among underground and normal metabolic reactions to form new metabolic pathways in a single step. We are investigating the evolution of new metabolic pathways that utilize NSAR activity to understand the combined contributions of horizontal gene transfer and underground metabolism. Our initial comparative genomics studies show that NSAR genes, which have been horizontally transferred to many different microbial species, have been integrated into various genome contexts. This suggests that new pathways that utilize NSAR enzymes have evolved multiple times. We are using molecular phylogenetics to trace the origins of these metabolic pathways and experimentally characterizing the enzymes involved. Furthermore, we are using experimental evolution to investigate the de novo evolution of new metabolic pathways.

M. E. Glasner, D. P. Truong, B. C. Morse, How enzyme promiscuity and horizontal gene transfer contribute to metabolic innovation. FEBS J. 287, 1323–1342 (2020).