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AE Pilot Project

Anaerobic enzymology (AE) Pilot Project

Project period: 2014 – present

Squire J. Booker, Pennsylvania State University



◦    Radical-dependent enzymology allows the execution unusual chemical transformations (C-H bond functionalization, isomerization, sulfur insertion, C-O bond formation, anaerobic oxidation) via an iron-sulfur cluster cleaving S-Adenosyl methionine (SAM) and producing a radical intermediate or abstraction of a hydrogen from glycine producing a glycyl radical



◦    Traditional TIM barrel or ThiC-like TIM barrel or 10-stranded alpha/beta barrel “core”, with a diverse C-terminal domain


Challenges for Function Assignment

◦    wide range of chemical reactions carried out by members of the Radical SAM and Glycyl Radical Superfamilies

◦    many anaerobic members impedes adequate coverage of sequence space due to limitations in gene/protein acquisition

◦    the presence and participation of an Fe-S cluster provides a challenge in modeling efforts


Value to Integrated Strategy

◦    provides a scenario complementary to aerobic metabolic pathways by exploring adaptations that accommodate metabolism under anaerobic conditions

◦    serves as a test case for anaerobic protein production as well as anaerobic bacterial culturing

     ◦       provides an experimental platform for testing sequence/function relationships among highly homologous, yet functionally diverse targets



As the EFI shifts its focus to metabolic pathways in the gut microbiota, the decision was made to establish expertise in the anaerobic enzymology, in particular for discovery of novel functions in the radical SAM and glycyl radical superfamilies.  Dr. Squire Booker (Penn State) is an expert on the radical SAM superfamily; he played a major community liaison role in the radical SAM workshop that was held in September 2012.  The plan is to include Dr. Booker in the competing renewal application, with an “upgrade” of the Year 5 Anaerobic Enzymology Pilot Project to the Anaerobic Enzymology Bridging Project.


The Anaerobic Enzymology Pilot Project will focus on functional assignment of members of the radical SAM and glycyl radical enzyme superfamilies.  The proteins will be cloned, purified, and crystallized using the new anaerobic protein production and crystallization facility at AECOM.  The plan is that the Pilot Project will use SSNs and GNNs together with pathway docking by the Computation Core to discover new functions in these superfamilies.  The radical SAM superfamily is huge (now >160,000 members), the glycyl radical superfamily is more modest in size (now >20,000 members), so we anticipate that these superfamilies are fertile ground for discovering new functions/metabolic pathways important to gut human microbiome metabolism and physiology (including host interactions).


This Radical SAM Superfamily shares a common enzymatic reaction: an iron-sulfur cluster cleaving S-Adenosyl Methionine (SAM) and producing a radical intermediate. Radical-dependent enzymology allows the execution of a wide range of unusual chemical transformations (C-H bond functionalization, sulfur insertion, C-O bond formation, etc.), thus providing the EFI with a new sequence space that will prove to be rich with novel functions. Alternatively, the Glycyl Radical Superfamily produces a radical glycine on the main chain of the protein, but then also catalyzes a variety of functions related to nucleotide, pyruvate, and toluene metabolism. Importantly, Radical SAM and glycyl radical enzymes are often taking the place of enzymes that rely on the cleavage of molecular oxygen for production of potent oxidants - thus these enzymes play a large role in anaerobic metabolism and are accordingly abundant in the portion of the biosphere that lacks access to oxygen.


While the addition of the Anaerobic Enzymology Pilot Project provides our Bioinformatics and Computational Cores with the challenging task of evolving prediction algorithms to a new enzyme class, the origins of these enzymes, often anaerobic bacteria, will test the flexibility of our Protein, Structure and Microbiology Cores. In preparation, labs at both the Albert Einstein College of Medicine (AECOM) and the University of Illinois at Urbana-Champaign (UIUC) have acquired new anaerobic chambers. At AECOM, the Protein Core has recapitulated their entire high-throughput prokaryotic protein purification and crystallization infrastructure within an anaerobic chamber (i.e., glove box), so as to bring these capabilities to the study of oxygen sensitive macromolecules. In September 2013, Dr. James Love (AECOM) designed and commissioned a novel resource, which includes approximately 50 linear feet of glove box space. This anaerobic chamber houses all elements required for high throughput purification, crystallization and crystal mounting for subsequent X-ray structure determination, functional and mechanistic analysis. Nearly 900 miles away, the Microbiology Core (UIUC) has purchased a 2-person vinyl anaerobic chamber for installation into a Biosafety Level 2 facility for culturing constitutive and facultative anaerobes. The majority of these microbes are members of the human gut microbial community. Any functional characterizations conducted in vitro will be corroborated with in vivo growth phenotyping, transcriptomics, and metabolomics.


Selection of Radical SAM enzymes for input into the protein production pipeline is already underway. Sequence Similarity Networks (SSNs) are directing researchers toward divergent clusters of protein sequences where no functions have been experimentally verified. This mechanism of prioritization is anticipated to provide the highest likelihood of discovering novel functions in this protein superfamily. Of the 3700 targets identified from the Radical SAM superfamily, approximately 1000 have been successfully cloned and tested for small-scale expression, with actionable amounts of protein being produced for approximately 35% of clones. Proteins have been scaled up and purified successfully via automated methods, reconstituted to yield homogeneous Fe-S clusters, and protein crystals have been grown under strict oxygen free conditions. Concurrently, the Microbiology Core is bolstering the Protein Core’s repertoire by purifying additional genomic DNAs from anaerobic microbes such as Bacteroides spp. (Bacteroidetes), Clostridium spp. (Firmicutes), and Collinsella aerofaciens (Actinobacteria). The addition of these bacteria will not only increase the pipeline’s biological diversity, but also increase the human health relevance of pipeline results in the form of human gut microbiota-based functional discovery. All Cores look forward to this exciting expansion into anaerobic territory.


Representative References

·       Booker, Squire J., and Tyler L. Grove. “Mechanistic and functional versatility of radical SAM enzymes.” F1000 Biol. Rep. (2010), 2: 52.

·       Vey, Jessica L., and Catherine L. Drennan. “Structural Insights into Radical Generation by the Radical SAM Superfamily.” Chemical Reviews 111.4 (2011): 2487–2506.

·       Eklund, Hans and Marc Fontecase. “Glycyl radical enzymes: a conservative structural basis for radicals.” Structure (1999), 7: R257-R262.