The identification of novel enzymatic functions in the enolase superfamily

The correct identification of protein functions is of paramount importance in the Post-Genomic Era. The comparative ease and cost-effectiveness of obtaining large amounts of high-quality genome sequence information has resulted in an explosion in the scope of proteins in which to study. Though this is revolutionizing the fields of biochemistry and enzymology, the unfortunate drawback is that a majority of newly sequenced genes encode proteins which have unclear functions. In order advance the field of Genomic Enzymology to a level that is at least commensurate to that of Genomics, new strategies must be developed that utilize the specificity information encoded in the gene sequence to accurately predict a protein's function.;The mechanistically diverse enolase superfamily represents a fertile testing ground in the expansion of functional identification technologies. Due to the shared chemical and structural features inherent to this superfamily, a great deal of information is already known about members with unknown functions. These proteins can then be subjected to enzymological, structural and computational studies to gather details about the breadth of functional diversity within the superfamily. Based on homology and operon context analysis, it is estimated that less than fifty percent of the known members of the enolase superfamily have a correctly annotated function. Thus this superfamily represents a reasonable model system in the effort to develop the ability to identify novel protein function directly from gene sequences.;In this thesis, I describe the identification and characterization of three novel functions within the enolase superfamily, each of which represents both a specificity that had not been previously seen as well as a catalytic strategy that had also been so far unidentified. L-Rhamnonate dehydratase (RhamD) is a homologue of mandelate racemase (MR) but catalyzes the dehydration of a six-carbon acid-sugar substrate in a previously uncharacterized pathway for the catabolism of this compound. Structural and biochemical characterization of this enzyme reveals that it is promiscuous for the substrate it can dehydrate but also has evolved a catalytic strategy henceforth not seen in the MR subgroup.;D-Mannonate dehydratase (ManD) is a divergent member of the enolase superfamily which is involved in a seemingly redundant pathway for the metabolism of that compound. This enzyme utilizes both a catalytic and substrate-binding strategy previously unseen in the superfamily. An extended loop in the catalytic domain, unique to this enzyme family, possesses the first known example of a catalytic tyrosine in the superfamily and, unlike RhamD, ManD is highly specific for its substrate.;Galactarate dehydratase II (GalrD-II) is also a divergent member of the enolase superfamily. This enzyme is one of the most divergent members of the superfamily and utilizes one of the most unique catalytic strategies in the superfamily. Tyrosine side chains serve as both the base and acid catalysts for this enzyme's highly specific dehydration of the levorotary arm of the galactarate molecule. The study of this enzyme was a crucial stepping-stone towards the idealized world of ab initio functional prediction as an in silico screening of an unliganded crystal structure provided strong evidence about the nature of the substrate for this enzyme.;Studying the functional and catalytic diversity in the enolase superfamily is a valuable enterprise in accelerating the field of genomic enzymology. This is of paramount importance as the accumulation of genomic data is threatening to outstrip the ability for biochemists to study proteins at a pace that is commensurate with sequencing. Therefore, these contributions to defining the functional diversity within the confines of a mechanistically diverse superfamily are undeniably important in providing the basic understanding to allow for the most rapid possible assignment of protein functions.