Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

Abstract

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e −70 ) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.chain-elongation | prenyltransferase T he five-carbon molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are the fundamental building blocks for isoprenoid compounds. Beginning with DMAPP, a series of polyprenyl diphosphates with C 10 (geranyl diphosphate, GPP), C 15 (farnesyl diphosphate, FPP), C 20 (geranylgeranyl diphosphate, GGPP), C 25 (farnesylgeranyl diphosphate, FGPP), and higher molecular weight isoprenoid chains are synthesized by polyprenyl transferases (PTSs). With only a few exceptions, PTSs provide substrates for all but a few branch point enzymes for the biosynthesis of the more than 55,000 known isoprenoid metabolites, including monoterpenes, sesquiterpenes, diterpenes, sterols, carotenoids, ubiquinones, and prenylated proteins and peptides fulfilling essential roles in cells (Fig. S1) (1, 2).There are two distinct classes of PTSs, E-PTS forming trans bonds and Z-PTS forming cis bonds throughout chain elongation. The carbon skeletons of the great majority of isoprenoid metabolites are derived from products of E-PTSs, which share a common protein fold and two functionally important Asp-rich (DDXXD) motifs (1, 3, 4). E-PTSs synthesize linear allylic diphosphates ranging f...

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases

“…The five-carbon building block, isopentenyl diphosphate (IPP), 2 and its isomer, dimethylallyl diphosphate (DMAPP), are precursors for the synthesis of over 55,000 structurally and chemically diverse isoprenoids including dolichols, sterols, the side chain of ubiquinone and chlorophyll, prenyl groups of certain proteins and tRNA molecules, carotenoids, plant and animal steroid hormones, and aromatic compounds (1,2). In a majority of eukaryotic cells, Archaea, and some Eubacteria, IPP and DMAPP are synthesized via the mevalonate pathway (MVA).…”

“…trans-PTs share a common protein fold and two conserved, functionally important asparagine-rich (DDXX(XX)D) motifs. trans-PTs synthesize linear allylic diphosphates ranging in size from 2 (C 10 ) to 10 (C 50 ) isoprene units (1). The precise mechanisms of prenyl chain elongation are well established based on structural and sitedirected mutagenesis studies of a number of enzymes in this class (1,2,5).…”

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

“…Terpenoids and molecules containing terpenoid fragments are produced from two basic five-carbon building blocks: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1, 2). IPP and DMAPP are then converted into a series of terpenoid diphosphate esters of increasing chain lengths by chain length selective polyprenyl diphosphate synthases to give geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP, C 15 ), geranylgeranyl diphosphate (GGPP, C 20 ), and higher five-carbon homologs (3). These molecules are substrates for terpene synthases, which catalyze hydroxylation, elimination, cyclization, and rearrangement reactions to produce much of the structural diversity of terpenoid molecules found in nature (4,5).…”

“…One of the key objectives of the enzyme function initiative (EFI) is the development of methods that facilitate functional assignments for unknown terpene synthases as was previously demonstrated for the polyprenyl transferases (3,19). Quantum mechanics-only and quantum mechanics/molecular mechanics (QM/MM) calculations have provided important insights about the mechanisms of reactions catalyzed by terpene synthases (20)(21)(22)(23)(24).…”

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

“…In the case of the transprenyltransferases, which are structurally and mechanistically distinct from the cis-prenyltransferases, bulky aromatic residues at the interface between two a-helices are known to determine product chain length, and a combination of crystallography, structural modeling, bioinformatics, and biochemical analyses has led to the development and validation of predictive models of their functional properties (Ohnuma et al, 1996(Ohnuma et al, , 1997(Ohnuma et al, , 1998Tarshis et al, 1996;Wallrapp et al, 2013).…”