Amino Acid Acceptor Research Articles

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site.

Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-acceptor amino acid identification and ADPr-site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1-dependent in vivo ADPr-acceptor amino acid. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans-modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

Metabolites in visceral fat: useful signals of metabolic syndrome?

Metabolic syndrome comprises a series of health conditions, such as increased blood pressure, high blood sugar, excess abdominal fat, and altered circulating cholesterol or triglyceride levels. A fast growing number of affected individuals are at an increased risk of heart disease, stroke and type-2 diabetes. Obesity, especially build-up of visceral fat, is a recognized major risk factor for the development of metabolic syndrome. However, our understanding of the mechanistic links and biomarkers that associate visceral fat with the development of conditions underlying metabolic syndrome is still inadequate. In a recent paper published in the Biochemical Journal [Biochem. J. (2018) 475, 1019–1035], Candi et al. address this lack of knowledge, performing high-throughput metabolomics analysis of visceral fat isolated from obese individuals, with and without metabolic syndrome, and non-obese healthy controls. The authors identify alterations in metabolic pathways that distinguish pathologically from healthy obese subjects. They identify metabolic cues that point to oxidative and inflammatory burden as the leitmotifs of metabolic syndrome. Of particular interest is the identification of increased metabolism of γ-glutamyl amino acids and plasmalogens in pathological obesity. γ-glutamyl amino acids, generated through the transfer of a γ-glutamyl moiety from glutathione to an amino acid acceptor, are involved in glutathione metabolism and the response to oxidative stress, whereas plasmalogens, a poorly studied class of phospholipids, are known contributors to insulin resistance and hypertension. Both classes of metabolites are intriguing candidate biomarkers that warrant further investigation.

Evidences on the role of the lid loop of γ-glutamyltransferases (GGT) in substrate selection

γ-Glutamyltransferase (GGT) catalyzes the transfer of the γ-glutamyl moiety from a donor substrate such as glutathione to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a γ-glutamyl enzyme intermediate.The vast majority of the known GGTs has a short sequence covering the glutamate binding site, called lid-loop. Although being conserved enzymes, both B. subtilis GGT and the related enzyme CapD from B. anthracis lack the lid loop and, differently from other GGTs, both accept poly-γ-glutamic acid (γ-PGA) as a substrate. Starting from this observation, in this work the activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT. Results indicate that the absence of the lid loop seems not to be the sole structural feature responsible for the recognition of a polymeric substrate by GGTs. Nevertheless, time course of hydrolysis reactions carried out using oligo-γ-glutamyl glutamines as substrates showed that the lid loop acts as a gating structure, allowing the preferential selection of the small glutamine with respect to the oligomeric substrates. In this respect, the mutant B. subtilis GGT revealed to be more similar to E. coli GGT than to its wild-type counterpart. In addition, the transpeptidase activity of the newly produced mutant enzyme revealed to be higher with respect to that of both E. coli and wild-type B. subtilis GGT. These findings can be helpful in selecting GGTs intended as biocatalysts for preparative purposes as well as in designing mutant enzymes with improved transpeptidase activity.

β-L-Arabinofuranosylation Conducted by 5-O-(2-pyridinecarbonyl)-L-arabinofuranosyl Trichloroacetimidate

We describe a β-L-arabinofuranosylation method by employing the 5-O-(2-pyridinecarbonyl)-L-arabinofuranosyl trichloroacetimidate 10 as a donor. This approach allows a wide range of acceptor substrates, especially amino acid acceptors, to be used. Stereoselective synthesis of β-(1,4)-L-arabinofuranosyl-(2S, 4R)-4-hydroxy-L-proline (β-L-Araf-L-Hyp4) and its dimer is achieved readily by this method. Both the stereoselectivities and yields of the reactions are excellent. To demonstrate the utility of this methodology, the preparation of a trisaccharide in a one-pot manner was carried out.

Rational Engineering of a Flavoprotein Oxidase for Improved Direct Oxidation of Alcohols to Carboxylic Acids.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

Gene Tree Discordance Does Not Explain Away the Temporal Decline of Convergence in Mammalian Protein Sequence Evolution.

Several authors reported lower frequencies of protein sequence convergence between more distantly related evolutionary lineages and attributed this trend to epistasis, which renders the acceptable amino acids at a site more different and convergence less likely in more divergent lineages. A recent primate study, however, suggested that this trend is at least partially and potentially entirely an artifact of gene tree discordance (GTD). Here, we demonstrate in a genome-wide data set from 17 mammals that the temporal trend remains (1) upon the control of the GTD level, (2) in genes whose genealogies are concordant with the species tree, and (3) for convergent changes, which are extremely unlikely to be caused by GTD. Similar results are observed in a comparable data set of 12 fruit flies in some but not all of these tests. We conclude that, at least in some cases, the temporal decline of convergence is genuine, reflecting an impact of epistasis on protein evolution.

Bacterial Labionin-Containing Peptides and Sactibiotics: Unusual Types of Antimicrobial Peptides with Potential Use in Clinical Settings (a Review).

One of the biggest challenges faced presently by clinicians is the emergence of multidrug -resistant pathogens that can infect humans and animals. To control the infections caused by such pathogens the development of new drugs is required. Bacteria are a rich source of ribosomally -synthesized antimicrobial peptides known as bacteriocins, which are characterized by the presence of a self-defense immunity system. Labionin-containing lantibiotics and sactibiotics are posttranslationally modified bacteriocins with peculiar features. Labionin-containing peptides belong to subclass Ic lantibiotics in which the carbacyclic triamino triacid labionin, a structural variant of lanthionine, and a methyl-substitute labionin derivative are found, giving the molecule a labyrinthine structure. Sactibiotics are circular or linear peptides belonging to a distinct bacteriocin class (class V) which is characterized by the presence of cross-linkages formed by the thiol group of cysteine residues and the α-carbon of acceptor amino acids. A few examples of these bacteriocins have been described in the literature to date, although putative gene clusters with the potential to encode such peptides can be found in the genome of many bacterial species. Some peptides already under study exhibit potential biotechnological applications because of their remarkable antibacterial or antiviral activities, as well as their analgesic activity. Therefore, in this review, the main findings concerning these peptides will be addressed and discussed, with an emphasis on their potential use in clinical settings.

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism.

1α,25-Dihydroxyvitamin D3 (1,25-D3) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D3 on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D3 stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active de novo protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D3 did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D3 stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.

Towards understanding the crosstalk between protein post-translational modifications: Homo- and heterotypic PTM pair distances on protein surfaces are not random.

Post-translational modifications (PTMs) represent an important regulatory layer influencing the structure and function of proteins. With broader availability of experimental information on the occurrences of different PTM types, the investigation of a potential "crosstalk" between different PTM types and combinatorial effects have moved into the research focus. Hypothesizing that relevant interferences between different PTM types and sites may become apparent when investigating their mutual physical distances, we performed a systematic survey of pairwise homo- and heterotypic distances of seven frequent PTM types considering their sequence and spatial distances in resolved protein structures. We found that actual PTM site distance distributions differ from random distributions with most PTM type pairs exhibiting larger than expected distances with the exception of homotypic phosphorylation site distances and distances between phosphorylation and ubiquitination sites that were found to be closer than expected by chance. Random reference distributions considering canonical acceptor amino acid residues only were found to be shifted to larger distances compared to distances between any amino acid residue type indicating an underlying tendency of PTM-amenable residue types to be further apart than randomly expected. Distance distributions based on sequence separations were found largely consistent with their spatial counterparts suggesting a primary role of sequence-based pairwise PTM-location encoding rather than folding-mediated effects. Our analysis provides a systematic and comprehensive overview of the characteristics of pairwise PTM site distances on proteins and reveals that, predominantly, PTM sites tend to avoid close proximity with the potential implication that an independent attachment or removal of PTMs remains possible. Proteins 2016; 85:78-92. © 2016 Wiley Periodicals, Inc.

Transfer RNAs with novel cloverleaf structures.

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro.

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

Synthesis, photophysical properties of triazolyl-donor/acceptor chromophores decorated unnatural amino acids: Incorporation of a pair into Leu-enkephalin peptide and application of triazolylperylene amino acid in sensing BSA

The research in the field of design and synthesis of unnatural amino acids is growing at a fast space for the increasing demand of proteins of potential therapeutics and many other diversified novel functional applications. Thus, we report herein the design and synthesis of microenvironment sensitive fluorescent triazolyl unnatural amino acids (UNAA) decorated with donor and/or acceptor aromatic chromophores via click chemistry. The synthesized fluorescent amino acids show interesting solvatochromic characteristic and/or intramolecular charge transfer (ICT) feature as is revealed from the UV–visible, fluorescence photophysical properties and DFT/TDDFT calculation. HOMO–LUMO distribution shows that the emissive states of some of the amino acids are characterized with more significant electron redistribution between the triazolyl moiety and the aromatic chromophores linked to it leading to modulated emission property. A pair of donor–acceptor amino acid shows interesting photophysical interaction property indicating a FRET quenching event. Furthermore, one of the amino acid, triazolyl-perylene amino acid, has been exploited for studying interaction with BSA and found that it is able to sense BSA with an enhancement of fluorescence intensity. Finally, we incorporated a pair of donor/acceptor amino acids into a Leu-enkephalin analogue pentapeptide which was found to adopt predominantly type II β-turn conformation. We envisage that our investigation is of importance for the development of new fluorescent donor–acceptor unnatural amino acids a pair of which can be exploited for generating fluorescent peptidomimetic probe of interesting photophysical property for applications in studying peptide–protein interaction.

Codon information value and codon transition-probability distributions in short-term evolution

To understand the way the Genetic Code and the physical–chemical properties of coded amino acids affect accepted amino acid substitutions in short-term protein evolution, taking into account only overall amino acid conservation, we consider an underlying codon-level model. This model employs codon pair-substitution frequencies from an empirical matrix in the literature, modified for single-base mutations only. Ordering the degenerated codons according to their codon information value (Volkenstein, 1979), we found that three-fold and most of four-fold degenerated codons, which have low codon values, were best fitted to rank-frequency distributions with constant failure rate (exponentials). In contrast, almost all two-fold degenerated codons, which have high codon values, were best fitted to rank-frequency distributions with variable failure rate (inverse power-laws). Six-fold degenerated codons are considered to be doubly assigned. The exceptional behavior of some codons, including non-degenerate codons, is discussed.

Formation of Kokumi-Enhancing γ-Glutamyl Dipeptides in Parmesan Cheese by Means of γ-Glutamyltransferase Activity and Stable Isotope Double-Labeling Studies.

Recently, γ-glutamyl dipeptides (γ-GPs) were found to be responsible for the attractive kokumi flavor of Parmesan cheese (PC). Quantitation of γ-GPs and their parent amino acids in 13-, 24-, and 30-month ripened PC samples by LC-MS/MS and stable isotope dilution analysis (SIDA), in-cheese (13)C-labeling studies, followed by analysis of the γ-glutamyl transferase (GGT) activity revealed γ-GPs to be generated most efficiently after 24 months of ripening by a GGT-catalyzed transfer of the γ-glutamyl moiety of L-glutamine onto various acceptor amino acids released upon casein proteolysis. Following the identification of milk as a potential GGT source in PC, the functionality of the milk's GGT to generate the target γ-GPs was validated by stable isotope double-labeling (SIDL) experiments. Therefore, raw and heat-treated milk samples were incubated with L-glutamine-[U-(13)C] and acceptor amino acids (X) and the hetero- (γ-Glu-[(13)C5]-X) and homotranspeptidation products (γ-Glu-Gln-[(13)C10]) were quantitated by LC-MS/MS-SIDA using γ-Glu-Ala-[(13)C3] as the internal standard. High GGT activity to generate the γ-GPs and preference for L-phenylalanine and L-methionine as acceptor amino acids were found in raw milk and milk samples heat-treated for 10 min up to a maximum of 65 °C. In comparison, GGT activity and SIDL studies performed with inoculated Lactobacillus strains, including Lactobacillus harbinensis and Lactobacillus casei identified in PC by means of 16S rRNA gene sequencing, did not show any significant GGT activity and unequivocally demonstrated unpasteurized cow's milk, rather than microorganisms, as a key factor in γ-glutamyl dipeptide generation in Parmesan cheese.

The Complete Mitochondrial Genome of two Tetragnatha Spiders (Araneae: Tetragnathidae): Severe Truncation of tRNAs and Novel Gene Rearrangements in Araneae.

Mitogenomes can provide information for phylogenetic analysis and evolutionary biology. The Araneae is one of the largest orders of Arachnida with great economic importance. In order to develop mitogenome data for this significant group, we determined the complete mitogenomes of two long jawed spiders Tetragnatha maxillosa and T. nitens and performed the comparative analysis with previously published spider mitogenomes. The circular mitogenomes are 14578 bp long with A+T content of 74.5% in T. maxillosa and 14639 bp long with A+T content of 74.3% in T. nitens, respectively. Both the mitogenomes contain a standard set of 37 genes and an A+T-rich region with the same gene orientation as the other spider mitogenomes, with the exception of the different gene order by the rearrangement of two tRNAs (trnW and trnG). Most of the tRNAs lose TΨC arm stems and have unpaired amino acid acceptor arms. As interesting features, both trnSAGN and trnSUCN lack the dihydrouracil (DHU) arm and long tandem repeat units are presented in the A+T-rich region of both the spider mitogenomes. The phylogenetic relationships of 23 spider mitogenomes based on the concatenated nucleotides sequences of 13 protein-coding genes indicated that the mitogenome sequences could be useful in resolving higher-level relationship of Araneae. The molecular information acquired from the results of this study should be very useful for future researches on mitogenomic evolution and genetic diversities in spiders.

Bacterial glycosyltransferase toxins.

Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

Nutrient acquisition across a dietary shift: fruit feeding butterflies crave amino acids, nectivores seek salt.

Evolutionary dietary shifts have major ecological consequences. One likely consequence is a change in nutrient limitation-some nutrients become more abundant in the diet, others become more scarce. Individuals' behavior should change accordingly to match this new limitation regime: they should seek out nutrients that are deficient in the new diet. We investigated the relationship between diet and responses to nutrients using adult Costa Rican butterflies with contrasting feeding habits, testing the hypothesis that animals will respond more positively to nutrients that are scarcer in their diets. Via literature searches and our own data, we showed that nitrogen and sodium are both at lower concentration in nectar than in fruit. We therefore assessed butterflies' acceptance of sodium and four nitrogenous compounds that ranged in complexity from inorganic nitrogen (ammonium chloride) to protein (albumin). We captured wild butterflies, offered them aqueous solutions of each substance, and recorded whether they accepted (drank) or rejected each substance. Support for our hypothesis was mixed. Across the sexes, frugivores were four times more likely to accept amino acids (hydrolyzed casein) than nectivores, in opposition to expectation. In males, nectivores accepted sodium almost three times more frequently than frugivores, supporting expectations. Together, these results suggest that in butterflies, becoming frugivorous is associated with an increased receptivity to amino acids and decreased receptivity to sodium. Nectivory and frugivory are widespread feeding strategies in organisms as diverse as insects, birds, and bats; our results suggest that these feeding strategies may put different pressures on how animals fulfill their nutritional requirements.

Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the Identification of ADP-Ribosylation Sites.

ADP-ribosylation of proteins alters their function or provides a scaffold for the recruitment of other proteins, thereby regulating several important cellular processes. Mono- or poly-ADP-ribosylation is catalyzed by different ADP-ribosyltransferases (ARTs) that have different subcellular localizations and modify different amino acid acceptor sites. However, our knowledge of ADP-ribosylated proteins and their acceptor amino acids is still limited due to the lack of suitable mass spectrometry (MS) tools. Here, we describe an MS approach for the detection of ADP-ribosylated peptides and identification of the ADP-ribose acceptor sites, combining higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) on an LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated peptides to target in a second LC-MS/MS analysis. The combination of HCD with ETD fragmentation gave a more comprehensive coverage of ADP-ribosylation sites than that with HCD alone. We successfully identified different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods may be applied to complex samples, allowing comprehensive identification of ADP-ribosylation acceptor sites.

TRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily.

TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2′-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2′-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

Are Convergent and Parallel Amino Acid Substitutions in Protein Evolution More Prevalent Than Neutral Expectations?

Convergent and parallel amino acid substitutions in protein evolution, collectively referred to as molecular convergence here, have small probabilities under neutral evolution. For this reason, molecular convergence is commonly viewed as evidence for similar adaptations of different species. The surge in the number of reports of molecular convergence in the last decade raises the intriguing question of whether molecular convergence occurs substantially more frequently than expected under neutral evolution. We here address this question using all one-to-one orthologous proteins encoded by the genomes of 12 fruit fly species and those encoded by 17 mammals. We found that the expected amount of molecular convergence varies greatly depending on the specific neutral substitution model assumed at each amino acid site and that the observed amount of molecular convergence is explainable by neutral models incorporating site-specific information of acceptable amino acids. Interestingly, the total number of convergent and parallel substitutions between two lineages, relative to the neutral expectation, decreases with the genetic distance between the two lineages, regardless of the model used in computing the neutral expectation. We hypothesize that this trend results from differences in the amino acids acceptable at a given site among different clades of a phylogeny, due to prevalent epistasis, and provide simulation as well as empirical evidence for this hypothesis. Together, our study finds no genomic evidence for higher-than-neutral levels of molecular convergence, but suggests the presence of abundant epistasis that decreases the likelihood of molecular convergence between distantly related lineages.