Structure-based activity prediction for an enzyme of unknown function

Abstract

With many genomes sequenced, a pressing challenge in biology is predicting the function of the proteins that the genes encode. When proteins are unrelated to others of known activity, bioinformatics inference for function becomes problematic. It would thus be useful to interrogate protein structures for function directly. Here, we predict the function of an enzyme of unknown activity, Tm0936 from Thermotoga maritima, by docking high-energy intermediate forms of thousands of candidate metabolites. The docking hit list was dominated by adenine analogues, which appeared to undergo C6-deamination. Four of these, including 5-methylthioadenosine and S-adenosylhomocysteine (SAH), were tested as substrates, and three had substantial catalytic rate constants (10(5) M(-1 )s(-1)). The X-ray crystal structure of the complex between Tm0936 and the product resulting from the deamination of SAH, S-inosylhomocysteine, was determined, and it corresponded closely to the predicted structure. The deaminated products can be further metabolized by T. maritima in a previously uncharacterized SAH degradation pathway. Structure-based docking with high-energy forms of potential substrates may be a useful tool to annotate enzymes for function.

Figure 1. Sample transformations of metabolites from their ground state structure into the high-energy intermediate forms that were used for docking

Transformations were computed according to the conserved reaction mechanism of amidohydrolases, a nucleophilic attack of a hydroxide at an electrophilic centre atom. Every transformable functional group for each molecule was processed independently. If the high-energy structure was chiral, all stereoisomers were calculated. Reactions catalysed by the amidohydrolases cytosine deaminase (CDA), adenosine deaminase (ADA), dihydroorotase (DHO), d-hydantoinase (HYD), isoaspartyl-d-dipeptidase (IAD), N-acetyl-d-glucosamine-6-phosphate deacetylase (NaGA) and phosphotriesterase (PTE) are shown.

Figure 2. Binding and conversion of MTA by Tm0936

a, Stereoview of MTA in its high-energy intermediate form docked into the active site of Tm0936. Oxygen atoms are coloured red; enzyme carbons, grey; ligand carbons, green; hydrogens, white; nitrogens, blue; sulphur, orange; and the metal ion, purple. The oxyanion, representing the nucleophilic hydroxyl, ion-pairs with the metal ion and His 228. The ammonia leaving group is placed between Glu 203 and Asp 279, at 3.2 Å and 2.9 Å, respectively, also interacting with Ser 283 (3.2 Å). The N1-nitrogen donates a hydrogen bond to Glu 203, whereas N3 accepts one from His 173 (2.5 Å and 2.9 Å). Ribose hydroxyls hydrogen bond to Glu 84 (2.8 Å and 2.9 Å). Adenosines larger than MTA, such as SAH, make additional interactions with more distal residues, such as Arg 136 and Arg 148. All figures were rendered using PyMOL (http://pymol.sourceforge.net). b, The deamination of MTA to MTI, a reaction catalysed by Tm0936.

Figure 3. Comparing the docking prediction and the crystallographic result

Superposition of the crystal structure of Tm0936 in complex with SIH (red) and the docking predicted structure of the high-energy intermediate of SAH (carbons in green). Enzyme carbons are coloured light blue, SAH and enzyme oxygen atoms are coloured red, nitrogens blue and sulphurs orange. The purple sphere represents the divalent metal ion. An F_O–F_C omit electron density map for SIH is shown, contoured at 4.1 σ. The structure was determined at 2.1 Å resolution.