Andrew M. Gulick, Ph.D.
Hauptman-Woodward Institute - Senior Research Scientist
Assistant Professor of Structural Biology, SUNY-Buffalo

EDUCATION
B.S., Biochemistry, Brown University, 1989
Ph.D., Experimental Oncology and Biochemistry
University of Wisconsin-Madison, 1994

MAILING ADDRESS:
Hauptman-Woodward
Medical Research Institute
700 Ellicott Street
Buffalo, NY 14203-1102

CONTACT INFORMATION:
Tel. 716-898-8619
Fax. 716-898-8660
E-mail. gulick@hwi.buffalo.edu

Research Interests

Crystallographic studies of non-ribosomal peptide synthetases. Structures of multi-domain enzymes and analysis of enzyme reaction mechanisms.

Crystallographic and Functional Studies of Adenylate-Forming Enzymes

Research in our lab is focused primarily on the use of X-ray crystallography as a tool to study the structure and function of enzymes that catalyze interesting reactions.  One such family of enzymes is the adenylate-forming family of enzymes that play multiple roles in primary and secondary metabolism. This family of enzymes contains three sub-families:  Acyl-CoA synthetases, the adenylation domains of the non-ribomsomal peptide synthetases, and firefly luciferases. These enzymes all catalyze two-step reactions in which the first step is an adenylation reaction between ATP and a carboxylate to form an acyl-adenylate. The enzymes differ in the second half-reaction; luciferase uses an oxidative decarboxylation to form an activated compound that decomposes to give off light, while the remaining two subfamilies transfer the acyl group to either CoA or a CoA-like cofactor to form a thioester.  We have determined the structures of two acyl-CoA synthetases bound to different ligands that trap the enzyme in the active conformations for either the first or second half-reaction.  These studies have allowed us to propose that members of this family of enzymes adopt two different conformations:  The enzymes first catalyze the initial adenylation step in one conformation. Then, to catalyze the second half-reaction, the ~130 residue C-terminal domain rotates by 140° to adopt a second conformation that is competent to catalyze the second half-reaction. This unusually large rotation is interesting in that it allows the enzymes to use a mobile domain to reconfigure a single active site.  Residues from two different faces of the C-terminal domain are contributed to the active site to catalyze the distinct half-reactions. This large domain rotation is particular interesting in the context of the NRPS adenylation domains described below. The crystal structures of the adenylate-forming enzymes have provided the foundation for a series of functional studies that allow us further test this Domain Alternation hypothesis.

Crystallographic Studies of Non-Ribosomal Peptide Synthetases

The  Non-Ribosomal Peptide Synthetases (NRPSs) are a family of enzymes that produce important bacterial and fungal peptides which can have antibiotic, anticancer, and other properties. Additionally, some NRPS products are small siderophores that bacteria secrete to chelate iron under limiting conditions. Unlike most biosynthetic processes in which individual enzymes catalyze the different steps of the full reaction, the syntheses catalyzed by the NRPS enzymes are often all catalyzed by a large, multi-domain enzyme. In this regard, the NRPSs are considered modular enzymes–for each amino acid of the final peptide, the NRPSs contain a complete module that includes all of the steps for the incorporation and, possibly, chemical modification of that residue. The three essential domains of an NRPS module are, first, an adenylation domain which activates an amino acid and transfers the residue to the CoA-like cofactor of a carrier domain, the second essential domain. The third essential domain is a condensation domain that forms a peptide bond between residues that were activated on the carrier domains of adjacent module.

While crystal structures of individual domains of NRPSs have been determined previously, we have recently determined the first crystal structure of a two-domain NRPS enzyme, the EntB protein from E. coli.  This enzyme contains a C-terminal carrier protein domain bound to a N-terminal domain that is involved in synthesis of an alternate residue incorporated into the final NRPS product.  This structure provided the background for further functional studies in which we have constructed mutations in the carrier protein domain and determined the effects of these mutations on the ability of the upstream adenylation domain to recognize this carrier protein domain.  We plan to  further examine the interactions of the adenylation and carrier domains to understand the rules that govern domain interaction in the NRPS enzymes.  These insights will be necessary for the engineering of NRPS systems to generate novel peptide products.