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Center for Nonlinear Studies |
ABSTRACT: Function Discoveries within the Amidohydrolase Superfamily Ricardo MartÃ-Arbona anf Frank M. Raushel Protein function discovery is a complicated task that has brought together Chemistry, Biochemistry, Biology and Bioinformatics among others. The amidohydrolase superfamily is a functionally diverse group of enzymes found in every organism sequenced to date and its members are often involved in essential metabolic conversions. Structural and functional genomics, X-ray crystal structures, molecular docking, high throughput screening and mechanistic enzymology are powerful tools for the prediction of substrates and the involvement of these proteins in metabolic pathways. Several proteins with unknown biological function were subjected to these studies with positive outcomes. The structural and functional genomic studies of the gene neighborhood of Pa5106 from P. Aeruginosa helped in its classification as a N-formimino-L-glutamate iminohydrolase (HutF). HutF acts in the deimination of the fourth intermediate in the histidine degradation pathway. An amino acid sequence alignment between Pa5106 and other members of the amidohydrolase superfamily suggested that the residues Glu-235, His-269, and Asp-320 are involved in substrate binding and deimination. Comparison of several X-ray crystal structures with a molecular model of Pa5106 allowed the construction of a binding model for the substrate. Enzymology studies provided valuable information for the proposal of a mechanism of deimination of N-formimino-L-glutamate by HutF. In a different case, the combination of molecular docking, structural and functional genomics, and high throughput screening, demonstrated that Tm0936 from T. maritima is involved in an uncharacterized variant of the degradation pathway for S-adenosyl-L-methionine. Tm0936 acts on the deamination of S-adenosyl-L-homocysteine to S-inosyl-L-homocysteine; it also deaminates thiomethyladenosine and adenosine. Tm0172 then hydrolyzes the homocysteine group from S-inosyl-L-homocysteine. A third example is a protein for D. radiodurans (DR) and its lonely homolog from T. thermophilus (TTC). These proteins lack any significant amino acid sequence identity to any other known function proteins but contain those residues identified to impart deamination capabilities to other members of the amidohydrolase superfamily. Structural and functional genomics showed no promising leads toward the function annotation. Molecular docking experiments on DR suggested the hydrolysis of heterocyclic rings containing one or more exocyclic amine groups, especially purines and pyrimidines. Using this information, an extensive library of compounds containing heterocyclic rings with exocyclic amines was tested for activity. S-adenosyl-L-homocysteine, thiomethyladenosine and adenosine showed significant hydrolysis; shockingly, these compounds are nowhere to be found on the molecular docking results. It is a reminder of how protein folding and movement can affect computational experimentation when a simple X-ray snap-shot is taken in consideration and reiterates the importance and effectiveness of interdisciplinary efforts in the discovery of biological function for proteins with unknown activity.
Host: William Hlavacek