RESEARCH

Overview:

The overarching goal of our research is to elucidate functions of bacterial enzymes that belong to the large Gcn5-related N-acetyltransferase (GNAT) family. To accomplish this, we use a combination of enzymology, biophysical, and structural biology techniques. Enzymes from this family have diverse functions, which include acylation of a variety of molecules such as polyamines, antibiotics, peptides, and proteins. These enzymes have been implicated in a vast array of cellular functions and represent a system that is ripe for discovering new insight into mechanisms of antibiotic and herbicide resistance, metabolic regulation, and disease progression. Since most bacterial GNATs remain uncharacterized or have undiscovered functions, there is much to be learned about this family of proteins.

 

Our main focus in the laboratory is to delve deep into the underlying mechanisms of how a conserved structural architecture is used by nature to meticulously catalyze diverse reactions. To do this, we investigate the functions of GNATs from a variety of pathogenic and nonpathogenic bacteria, including Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Acinetobacter and Sinorhizobium meliloti, among others. The hope is that our basic research will lay the foundation for a more in-depth understanding of this family of enzymes and their importance in bacterial metabolism and infectious diseases.

Vision:

The GNAT field is vast, and it takes many scientists from around the world to push our knowledge about this enzyme family forward. A snapshot of where I see my laboratory's contribution to the field is shown here:

Main projects in the laboratory:

1.

Study bacterial GNATs of unknown function and develop chemical tools to obtain crystal structures with ligands bound to the acceptor sites for improved computational predictions of protein function.

While the core structure of GNATs is conserved across members of the GNAT superfamily, the acceptor substrate binding site varies and dictates the type of substrate that becomes acylated. The more we know about the types of molecules that can bind in this site and how the amino acid composition/properties of this site contribute to specific functions, the more accurate our computational predictors of protein function will be. Since each bacterial genome encodes multiple GNATs with diverse functions, but not all bacteria contain the same types of GNATs, it is important to study a variety of GNATs across multiple bacteria to ensure functional potential is widely explored and accurately predicted.

Study polyamine N-acetyltransferases across all domains of life to determine why some are allosteric and others are not, why some adopt diverse oligomeric states, and why their structures and substrate specificity are not conserved within and across domains of life.

2.

Polyamines are polycationic molecules that control a variety of cellular processes. During times of stress in bacteria these molecules become acetylated. Although many bacterial polyamine acetyltransferases have been studied in the past, there is still a massive amount of unknown information regarding the diversity of polyamine acetyltransferases in bacteria. One example where much can be learned is the polyamine acetyltransferase SpeG, which adopts a unique dodecameric structure and exhibits allosteric behavior.

3.

Study bacterial GNATs with diverse functions to gain a greater understanding of how their specific amino acid residues contribute to catalysis and substrate specificity or promiscuity.

We previously developed a broad-substrate screen as a tool to explore possible functions of GNATs. We continue to use this screen as a starting point for characterizing GNATs of unknown function. Once we have identified a possible substrate for a previously uncharacterized enzyme, we further kinetically and structurally characterize it to determine how the enzyme works. We are currently exploring enzymes that modify phosphinothricin, aminoglycoside antibiotics, polymyxin antibiotics, or amino acids. By exploring GNATs of diverse functions, we are better positioned to gain a global understanding of key mechanisms of GNAT function.

San Francisco State University

Kuhn Laboratory

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