In a seminal paper showing the power of computational modeling and docking, AH superfamily member Tm0936 from Thermotoga maritima was functionally assigned as an S-adenosyl homocysteine deaminase. The work reported here builds on this discovery by targeting distant homologues of Tm0936 to further define the AH superfamily. Through an integrated approach combining bioinformatics, computation, and experimental biochemistry, functional predictions were made and experimentally validated for fifteen proteins. The homologues were found to deaminate a variety of adenosine analogues, allowing for provisional annotation of over 800 hundred additional sequences. The results provide an excellent example of the proficiency of this multidisciplinary approach for large-scale assignment of enzyme function.
A substantial challenge for genomic enzymology is the reliable annotation for proteins of unknown function. Described here is an interrogation of uncharacterized enzymes from the amidohydrolase superfamily using a structure-guided approach that integrates bioinformatics, computational biology, and molecular enzymology. Previously, Tm0936 from Thermotoga maritima was shown to catalyze the deamination of S-adenosylhomocysteine (SAH) to S-inosylhomocysteine (SIH). Homologues of Tm0936 homologues were identified, and substrate profiles were proposed by docking metabolites to modeled enzyme structures. These enzymes were predicted to deaminate analogues of adenosine including SAH, 5′-methylthioadenosine (MTA), adenosine (Ado), and 5′-deoxyadenosine (5′-dAdo). Fifteen of these proteins were purified to homogeneity, and the three-dimensional structures of three proteins were determined by X-ray diffraction methods. Enzyme assays supported the structure-based predictions and identified subgroups of enzymes with the capacity to deaminate various combinations of the adenosine analogues, including the first enzyme (Dvu1825) capable of deaminating 5′-dAdo. One subgroup of proteins, exemplified by Moth1224 from Moorella thermoacetica, deaminates guanine to xanthine, and another subgroup, exemplified by Avi5431 from Agrobacterium vitis S4, deaminates two oxidatively damaged forms of adenine: 2-oxoadenine and 8-oxoadenine. The sequence and structural basis of the observed substrate specificities were proposed, and the substrate profiles for 834 protein sequences were provisionally annotated. The results highlight the power of a multidisciplinary approach for annotating enzymes of unknown function.
Figure 1. Sequence similarity network for proteins related to Tm0936 from Thermotoga maritima. A node (dot) represents an enzyme from a bacterial species, and an edge (a connecting line) indicates that the two proteins are related by a BLAST E-value of 10–100 or better. Proteins sharing sequence similarity with Tm0936 cluster into apparent subgroups, and 12 of these have been arbitrarily numbered and color-coded based on the network diagram. Subgroups are predicted to be functionally similar, and representatives from each subgroup, denoted by the letters a–p, were selected for purification and functional characterization.
Figure 2. Active site of Tm0936. The crystal structure of Tm0936 (PDB id: 2PLM) in the presence of SIH (green) highlights the four residues (dark gray) that are important for substrate binding. Arg-148 and Arg-136 bind to the carboxylate moiety of SAH; His-173 and Glu-84 interact with N3 of the purine ring and the 2′, 3′ hydroxyls of the ribose moiety, respectively. Faded residues denote the six residues that bind the zinc or facilitate proton-transfer reactions during the catalytic transformations. These residues are conserved in all of the proteins depicted in Figure 1.
Figure 4. Comparison of the crystallographic and modeled structures of Cv1032. (A) The crystal structure of Cv1032 (PDB: 4F0S) in a catalytically productive state (white ribbon), the crystal structure of Cv1032 (PDB: 4F0R) in a catalytically unproductive state (cyan ribbon), and the homology model of Cv1032 based on the X-ray structure of Tm0936 (PDB: 2PLM) (yellow ribbon). The inosine bound in the active site of Cv1032 (PDB: 4F0S) is highlighted (white stick). (B) The crystal structure of inosine (white stick) in the active site of Cv1032 (transparent white stick) and the docking pose of adenosine (yellow stick) in the modeled active site (transparent green stick) composed of the same set of residues as the active site.
Reprinted with permission from the Journal of the American Chemical Society.
© 2013 American Chemical Society.