Research Projects

Nitrogen assimilation is a fundamental metabolic process present in every living organism. Glutamate dehydrogenase (GDH) is an essential enzyme for nitrogen assimilation in all living organisms as it catalyzes NAD+ or NADP-dependent conversion of alpha-ketoglutarate to L-glutamate in the presence of ammonia. This is a critical reversible reaction at the branchpoint of carbon and nitrogen cycles, and thus GDH plays an important regulatory role in cellular metabolism. GDHs function as an oligomer consisting of 2-6 subunits. Based on the monomeric molecular weight (MW), GDHs can be classified into two major classes: Small-GDH (S-GDH50, monomer MW = 50 kDa) and Large-GDH (L-GDH). L-GDHs are further classified into two structural subclasses: L-GDH180 with monomers of ~180 kDa and another L-GDH115 with monomers of ~115 kDa. Many prokaryotes and lower eukaryotes (fungi) are reported to have more than one GDH that catalyze the same reaction, either with different coenzyme specificities or at different points of growth stages based on the availability of certain metabolites. Sometimes, they are structurally distinct, i.e. S-GDH50 and L-GDH (L-GDH115 and L-GDH180) and have differential metabolic regulations. The physiological context of coexistence of multiple GDH isoforms in the same organism and their differential metabolic regulations are not clearly understood. S-GDH50s are extensively studied in terms of structure and functions, and are reported to adapt a hexameric form and are ubiquitously distributed. Despite various studies on multiple GDHs in last 50 years, the evolutionary significance of L-GDHs with respect to their limited distribution, structural differences, and coexistence with S-GDHs remains quite underexplored and demands thorough as well as systematic studies using multidisciplinary approaches. In contrast to S-GDHs and L-GDH180 (only one report on structure), the L-GDH115 subclass has no structural information to date; and biochemical and cellular studies are also almost completely lacking. These gaps in current research undoubtedly provide a fantastic opportunity for deeper exploration on L-GDH115 and also raise three crucial questions: (a) What are the major differences in the structural architecture of S-GDHs and L-GDHs? (b) How are the different domains and their dynamics associated with the various metabolic regulations in L-GDHs? (c) What is the physiological significance of the coexistence of both S-GDH and L-GDH in the same organism? Saccharomyces cerevisiae expresses multiple isoforms of GDH belonging to different structural subclasses: S-GDH50 and L-GDH115, with different coenzyme specificities and differential expression levels during cellular growth based on the physiological needs. Hence, studying S. cerevisiae GDHs (ScGDHs) appears as an ideal system to obtain answers of the above questions for GDHs from various organisms. Extensive structural characterizations of L-GDH115 from S. cerevisiae (ScGDH2) using cryo-electron microscopy (Cryo-EM) and X-ray crystallography will be performed. Thorough biochemical and biophysical characterizations will be carried out to understand the enzyme kinetics and substrate/coenzyme/metabolite specificities of L-GDH115. Systematic studies including specific gene knockout experiments and phenotypic assays will be performed to understand the involvement of GDHs in regulating nitrogen assimilation under physiological conditions and cellular growth. Eventually, the integration of biochemical, structural, and physiological studies on L-GDHs will allow us to decipher how these enzymes emerged in response to physiological demands, their synergistic coexistence with other GDHs, and how their structure-function relationship evolved in course of time.