John A Gerlt

3107 IGB
Office: (217) 244-7414
Lab: (217) 333-2699
Fax: (217) 244-6538

Mail to: Department of Biochemistry
University of Illinois
600 S Mathews Ave
Urbana, IL 61801
Lab Page
Curriculum Vita

John A Gerlt

Professor of Biochemistry
Gutgsell Chair
Professor of Chemistry
Professor of Biophysics and Computational Biology

Research Topics

Biofuels, Bioinformatics, Enzymology, Genomics, Microbial Physiology, Molecular Evolution, Protein Dynamics, Protein Structure


B.S. 1969 Michigan State University
A.M. 1970; Ph.D. 1974 Harvard University
Postdoc. 1974-75, National Institutes of Health

Mechanisms of enzyme-catalyzed reactions, functional genomics, lignin deconstruction for biofuel production


With the availability of complete sequences for the genomes of numerous eubacteria, archaea, and eukaryotes, parallel structure/function studies of enzymes derived from a common progenitor is the best strategy for elucidating structure/function relationships for enzyme-catalyzed reactions. This approach, “genomic enzymology”, allows an efficient and precise identification of essential structure/function relationships that are important in catalysis. It also allows an understanding of the strategies used by Nature to evolve "new" enzymes and, therefore, provides design principles for the in vitro design of novel enzymes that catalyzed unnatural reactions. Our studies are enhancing the ability to predict the functions of "unknown" proteins discovered in genome projects.

We are studying three superfamilies of enzymes that are derived from common ancestors that share the ubiquitous (β/α)8-barrel fold. The members of the enolase superfamily catalyze different overall reactions initiated by abstraction of the α-proton of a carboxylate anion to form an enediolate anion intermediate that must be stabilized by the active site. The members of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) superfamily. The members of the orotidine 5'-monophosphate (OMP) decarboxylase suprafamily catalyze different reactions that do not share any mechanistic features; our discovery of this suprafamily supports the hypothesis that Nature is opportunistic and can both identify and utilize functionally versatile active site templates in the evolution of new enzymatic activities.

We also are discovering and characterizing novel enzymes involved in the degradation of lignin in plant biomass. The production of biofuels from plant biomass is limited by the access of degradative enzymes to the cellulose by lignin, a complex polymer of phenylpropanoid units. Identification and characterization of these enzymes will facilitate the design of new microbial species that can be used to improve the efficiency of biofuel production.


The active sites of members of the enolase superfamily contain a conserved binding site for a catalytically essential Mg2+. The acid/base groups are located on the rims of the active sites "cups" located at the C-terminal ends of (β/α)8-barrel domains, with one functional group located at the end of each of the eight β-strands.

We have identified >20 functions within the enolase superfamily, each of which is initiated by abstraction of the α-proton of a carboxylate to generate an enolate anion that is stabilized by the Mg2+; many of the substrates are acid sugars or dipeptides. Members of the superfamily often are functionally promiscuous, i.e., in addition to their natural reaction they also catalyze an "accidental" reaction that may be exploited for the evolution of a new activity. Understanding how conserved structure delivers different functions has allowed us to 1) predict the functions of unknown homologues in the sequence databases; and 2) redesign "old" enzymes to catalyze "new" reactions.


The members of the RuBisCO superfamily that catalyze abstraction of the α-proton of a ketone to generate an enolate that is stablized by an essential Mg2+. Most members of the superfamily catalyze the carboxylation of D-ribulose 1,5-bisphosphate to yield two molecules of 2-phosphoglyerate. However, genome sequencing projects have allowed identification of proteins that lack the active site residues required for CO2 fixation; these are called “RuBisCO-like proteins” or RLPs.

We have established structure/function relationships for a family of RLPs that catalyzes a tautomerization reaction in the methionine salvage pathway and, also, identified a novel isomerization reaction involving an intermediate in methionine salvage that likely participates in a novel, uncharacterized salvage pathway. Our efforts are focused on establishing structure/function relationships for members of the superfamily as well as deciphering the metabolic pathways in which they participate.


OMP decarboxylase, the penultimate step in pyrimidine nucleotide biosynthesis, is one of Nature’s most efficient catalysts. The decarboxylation reaction involves the metal ion independent formation of a vinyl carbanion intermediate. We are studying both the mechanism of the reaction, with our focus on understanding how the active site stabilizes the carbanion intermediate so that it can be kinetically competent. We also are studying the structural mechanism by which the “intrinsic binding energy” associated with the 5’-phosphate group of the substrate is used to enhance catalysis.

The OMP decarboxylase suprafamily includes not only OMP decarboxylase but also 3-keto-L-gulonate 6-phosphate decarboxylase and D-arabino-hex-3-ulose 6-phosphate synthase. In contrast to OMP decarboxylase, both utilize Mg2+ to stabilize an enolate anion intermediate. The active site residues are remarkably conserved in the suprafamily, although the mechanisms of the reactions are not conserved. The growing sequence databases now appear to contain novel members of the suprafamily that likely catalyze “new” reactions; the discovery of these reactions is under investigation.


As the sequence databases expand (now, >11,000,000 unique protein sequences are contained in the TrEMBL database), we are wanting to identify members of all three superfamilies that have unknown functions--these are determined in genome projects without regard to biological and biochemical function. Indeed, functional assignment of proteins of unknown function is a major challenge in postgenomic biology that is limiting both understanding the scope of Nature’s diversity and, also, exploiting that diversity for biomedical and industrial applications.

To address this problem, we are leading two NIH funded projects that are developing an integrated sequence/structure based strategy to predict the substrates (and, therefore, functions) of unknown members of functionally diverse enzyme superfamilies that are discovered in genome projects. In these multidisciplinary projects, experts in functional biology (mechanistic enzymology), structural biology (X-ray crystallography), computational biology (bioinformatics, modeling, and in silico ligand docking), and microbiology (genetics, transcriptomics, and metabolomics) are brought together to address the problem of functional assignment of unknown proteins. This multidisciplinary approach reflects the intellectual and practical demands of assigning functions to unknown/uncharacterized enzymes.

A Program Project (P01GM071790, “Deciphering Enzyme Specificity”) is focused on the functional diverse enolase and amidohydrolase (Frank Raushel, Texas A&M) superfamilies. A new Glue Grant (U54GM093342, “Enzyme Function Initiative”) is additionally focused on the functionally diverse glutathione transferase (Richard Armstrong, Vanderbilt University School of Medicine), haloalkanoic acid dehalogenase (Karen Allen, Boston University; Debra Dunaway Mariano, New Mexico), and isoprenoid synthase (Dale Poulter, Utah) superfamilies. Protein production and structure determination is lead by Steve Almo (Albert Einstein College of Medicine). Collaborators at UCSF provide expertise in bioinformatics (Patsy Babbitt) and homology modeling/in silico ligand docking (Matt Jacobson, Andrej Sali, and Brian Shoichet). And, John Cronan (genetics) and Jonathan Sweedler (metabolomics) at Illinois are investigating the physiological roles of novel enzymatic activities that are discovered by the in silico and enzymological approaches.


The polymeric carbohydrates in plant biomass that are the raw materials for biofuels are contained in a complex matrix. The matrix includes not only crystalline cellulose that is a polymer of glucose (the primary source of biofuels) but also both hemicellulose (a complex, heterogeneous polymer of several carbohydrates) and lignin. Hemicellulose and lignin, together “lignocellulose”, prevent access of cellulolytic enzymes to the cellulose, thereby decreasing the efficiency of biofuel production. Industrial approaches for the degradation of lignocellulose have been devised, e.g., treatment with acid, alkali, and/or heat, but these are not particularly efficient or environmentally friendly. The goal of our program is to devise microbiological and biochemical approaches for degradation of lignin so that plant biomass can be more efficiently converted to fuels.

Lignin, a complex polymer of phenylpropanoid units, has many types of chemical linkages that provide not only mechanical stability but also resistance to chemical degradation. Because many of the linkages involve stable aromatic structures, drastic nonenzymatic procedures have been used for depolymerization. Strains of Streptomyces have been reported to depolymerize lignin; however, the oxidative and hydrolytic enzymes involved in this process have not been identified. We are focused on discovering and characterizing these novel enzymes because they may provide a more efficient and environmentally friendly approach for lignin depolymerization.

The objectives of this program are to 1) to use genome sequences to discover degradative pathways for lignin; 2) mechanistically and structurally characterize the enzymes in those pathways; and 3) design new metabolic pathways so that lignin degradation can be facilitated and biofuel production enhanced.

This program is supported by the Energy Biosciences Institute (funded by BP) and also involves John Cronan (Microbiology; microbial physiology and genetics) and Satish Nair (Biochemistry; x-ray structure determination).

Representative Publications

Divergent Evolution in the Enolase Superfamily: Strategies for Assigning Functions, J. A. GERLT, P. C. Babbitt, M. P. Jacobson, and S. C. Almo, J. Biol. Chem. 2012, 287, 29-34.

Homology Models Guide Discovery of Diverse Enzyme Specificities Among Dipeptide Epimerases in the Enolase Superfamily, T. Lukk. A. Sakai, C. Kalyanaraman, S. D. Brown, H. J. Imker, L. Song, A. A. Fedorov, E. V. Fedorov, R. Toro, B. Hillerich, R. Seidel, Y. Patsklvsky, M. V. Vetting, S. K. Nair, P. C. Babbitt, S. C. Almo, J. A. GERLT, and M. P. Jacobson, Proc. Nat. Acad. Sci USA 2012, 109, 4122-4127.

OMP Decarboxylase: Phosphodianion Binding Energy is Used to Stabilize a Vinyl Carbanion Interrmediate, B. Goryanova, T. L. Amyes, J. A. Gerlt, and J. P. Richard, J. Amer. Chem. Soc. 2011, 133, 6545-6548.

Mechanism of the Orotidine 5’-Monophosphate Decarboxylase Reaction: Importance of Residues in the Orotate Binding Site, V. Iiams, B. J. Desai, A. A. Fedorov, E. V. Fedorov, S. C. Almo, and J. A. GERLT, Biochemistry 2011, 50, 8497-8507.

The Enzyme Function Initiative, J. A. GERLT, K. N. Allen, S. C. Almo, R. N. Armstrong, P. C. Babbitt, J. E. Cronan, D. Dunaway Mariano, H. J. Imker, M. P. Jacobson, W. Minor, C. D. Poulter, F. M. Raushel, A. Sali, B. K. Shoichet, and J. V. Sweedler, Biochemistry 2011, 50, 9950-9962.

Activation of R235A Mutant Orotidine 5'-Monophosphate Decarboxylase by the Guanidinium Cation: Effective Molarity of the Cationic Side Chain of Arg-235, S. A. Barnett, T. L. Amyes, B. M. Wood, J. A. Gerlt, and J P. Richard, Biochemistry 2010, 49, 824-826.

Conformational Changes in Orotidine 5’-Monophosphate Decarboxylase: “Remote” Residues that Stabilize the Active Conformation, B. M. Wood, T. L. Amyes, A. A. Fedorov, E. V. Fedorov, A. Shabila, S. C. Almo, J. P. Richard, and J. A. Gerlt, Biochemistry 2010, 49, 3514-3516.

Product Deuterium Isotope Effects for Orotidine 5’ Monophosphate Decarboxylase: Effect of Changing Substrate and Enzyme Structure on the Partitioning of the Vinyl Carbanion Reaction Intermediate, K. Toth, T. L. Amyes, B. M. Wood, K. Chan, J. A. Gerlt, and J. P. Richard, J. Amer. Chem. Soc. 2010, 132, 7018-7024.

Enzyme (Re)Design: Lessons from Natural Evolution and Computation, J. A. GERLT and P. C. Babbitt, Curr. Opin. Chem. Biol. 2009, 13, 10-18.

Mechanism of the Orotidine 5’ Monophosphate Decarboxylase Catalyzed Reaction: Effect of Solvent Viscosity on Kinetic Constants, B. M. Wood, K. K. Chan, T. L. Amyes, J. P. Richard and J A. GERLT, Biochemistry, 2009, 48, 5510-5517.

Mechanism of the Orotidine 5’ Monophosphate Decarboxylase Catalyzed Reaction: Evidence for Substrate Destabilization, K. K. Chan, B. McKay Wood, A. A. Fedorov, E. V. Fedorov, T. L. Amyes, J. P. Richard, S. C. Almo, and J. A. GERLT, Biochemistry, 2009, 48, 5518-5531.

Acetoacetate Decarboxylase: Hydrophobics, Not Electrostatics (News & Views), J. A. GERLT, Nature Chem. Biol. 2009, 5, 454-455.

The Relationship between Active Site Loop Size and Thermodynamic Activation Parameters for Orotidine 5’-Monophosphate Decarboxylase from Mesophilic and Thermophilic Organisms, K. Toth, T. L. Amyes, B. M. Wood, K. K. Chan, J. A. GERLT, and J. P. Richard, Biochemistry 2009, 48, 8006-8013.

Computation-Facilitated Assignment of Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacililus iheyensis, J. R. Rakus, C. Kalyanaraman, A. A. Fedorov, E. V. Fedorov, F. P. Mills Groninger, R. Toro, J. Bonanno, K. Bain, J. M. Sauder, S. K. Burley, S. C. Almo, M. P. Jacobson, and J. A. GERLT, Biochemistry 2009, 48, 11546-11558.

Complete Publications List