Hausinger 96-03520 Hausinger Part 1. Technical This study uses 2,4-dichlorophenoxyacetic acid (2,4-D)/alpha-keto glutarate dependent dioxygenase (tfdA) from Alcaligenes eutrophus as a model protein to examine the metallocenter properties and enzyme mechanisms in catalysis. This enzymes catalyzes the first step in biodegradation of the broadleaf herbicide 2,4-D. It couples the hydroxylation of 2,4-D, forming a compound that spontaneously decomposes to yield glyoxylate and 2,4-dichlorophenol, with the conversion of alpha-KG to CO2 and succinate in a ferrous ion-dependent reaction. TfdA is especially well suited to serve as a paradigm of this enzyme class because it is relatively small, highly soluble, end readily available in large quantities by using recombinant cell extracts and a simple two-step purification protocol. Furthermore, the tfdA gene is easily manipulated by genetic and recombinant methods. The objectives and approaches are: (1) characterize the metallocenter in TfdA bond to Fe(Il) or substituted metals by spectroscopic methods such as , X-ray absorption, and electron paramagnetic resonance ; (2) Identify the residues that participate as metallocenter or in substrate or cofactor binding and enzymatic catalysis by site-directed mutagenesis; (3) define the enzyme mechanism by examining the reactivity of TfdA towards alternate substrates, inhibitors, and inactivators; and (4) initiate studies on a related protein from E. coli, TauD, a sulfonate hydroxylase that degrades sulfonates to form sulfite, the cellular sulfur source. Results from these studies, designed to further the understanding of the novel biodegradative chemistry of TfdA, and other enzymes, may be useful to biotechnological and bioremediative applications such as the selective hydroxylation or decomposition of other compounds. Part 2. Non-technical This project seeks to characterize the enzymatic mechanism of TfdA, an enzyme that catalyzes the first step in the decomposition of the broadleaf herbicide 2,4D (2,4-d ichlorophenoxyacetic acid), from Alcaligenes eutrophus. TfdA is a representative of an important, yet poorly understood, class of ferrous ion-dependent enzymes that couple the oxygen-requiring decomposition of alpha-ketoglutarate to the hydroxylation of a substrate. Changes occurring at the metallocenter will be examined by spectroscopy, the roles of selected amino acid residues studied by molecular biological methods and the enzyme mechanism deduced by analyzing its reactivity towards substrates, inhibitors, and inactivators. In addition, the properties of this enzyme will be compared to those proteins related in sequence. Results from these studies should advance the understanding of the novel reaction mechanisms of enzymes useful in biotechnology and biodegradation. 80 Fane The proper assembly of viral proteins and nucleic acids into a biologically active virion involves numerous and diverse macromolecular interactions. The main objectives are to elucidate these critical interactions and to define the structural domains of the responsible macromolecules. A combination of genetic, biochemical and structural approaches (X-ray crystallography) will be employed to accomplish the object. Like molecular chaperones, scaffolding proteins direct other proteins in achieving their proper three-dimensional conformations. Within the context of this analogy, the atomic structure of a procapsid offers a depiction of a chaperone-like protein complexed with its substrate. Prior results suggest that the Microviridae internal scaffolding proteins, gpB, share many properties with molecular chaperones. Prior results also indicate that the internal scaffolding proteins either possess inherent flexibility or interact with their substrates in nonspecific manners, perhaps via interfaces. Determining the atomic structures of hybrid procapsids, containing foreign scaffolding proteins, will directly address this questio n. The present (X174 procapsid structure does not contain the internal scaffolding protein which is lost during purification. During the first year of support, alternate genetic and purification strategies will be explored to stabilize this protein. The current protocols will still be employed to gather additional data to refine the external scaffolding protein structure. Also within the first year, the genetic and purification strategies needed to generate alpha procapsids will be developed. Continuation of the structural work, done in collaboration with Dr. M. G. Rossmann, will proceed throughout the support period. The recently solved atomic structure of the external scaffolding protein, gpD, suggests that it also shares many features with molecular chaperones. Thus gpD, like a molecular chaperone, can bind to other proteins in many different ways. While some domains make contact with the coat protein in varied manners, one of these domains may determine the protein's substrate specificity for a particular viral coat protein. the plasmid-based cross complementation system has been extended to this gene. Unlike the internal scaffolding proteins which exhibit a great deal of divergence in primary structure, the external scaffolding proteins share 75% sequence identity. The (X174 protein, however, is unable to productively direct the assembly of other Microviridae virions. A comparison of the primary structure reveals that the divergent residues are localized to the NH2 -termini of the proteins which forms a large (-helix in the (X174 atomic structure. These observations suggest a model in which the scaffolding's specificity for a particular viral coat protein resides in this region. This hypothesis will be tested with chimeric polypeptides. During the first year of support, restriction sites needed to construct chimeric external scaffolding proteins will be introduced. The (X174 gene will be recloned and clones of other Microviridae D genes will be generated. Characterization of the chimeric genes and gene products will commence in the second year. The results of these analyses may also provide insights into the design of recombinant proteins. All viruses must assemble themselves by means of multiple protein interactions. viral assembly is often dependent on proteins known as scaffolding proteins. Analogous to scaffoldings used in the construction of buildings, scaffolding proteins are found in virus assembly intermediates but not in the mature viruses. Two different scaffolding proteins, external and internal, are required for the assembly of the Microviridae family of viruses. With These viruses we are able to purify viral intermediates which still include the external scaffolding protein. By examining the atomic structure of the external scaffolding protein and performing genetic analyses with both the internal and external proteins, we have determined that different regions of the scaffolding proteins may have specific and identifiable functions. Refining the atomic structure of these proteins and testing our hypotheses regarding their various functions by constructing hybrid Microviridae scaffolding proteins are the main objectives of the proposed work. The results of these analyses will provide further insights into virus assembly and the design of recombinant proteins.
