Prey-specialised spiders often do not have brood care and may not deposit eggs in the proximity of the preferred prey. Thus, naïve spiderlings are left to their own to find their focal prey. Our aim was to reveal whether the choice of a specific prey is innate and whether familiarisation with a certain prey will condition prey choice. We used the myrmecophagous spider Euryopis episinoides, which specialises on Messor ants. It finds ants using chemical cues deposited on the substrate. Naïve spiderlings were offered chemical cues from Messor and Myrmica ants and Drosophila flies. They chose significantly more chemical cues from Messor ants than those from Drosophila flies. Then spiderlings were assigned to three prey treatments: fed with Messor ants only (optimal prey), fed with Myrmica ants only (suboptimal prey) or fed with Drosophila flies only (detrimental prey) until adulthood. Every 2 weeks, all spiders from all treatments were offered chemical cues from the three prey types and the frequency of choice and latency to assuming a posture were recorded. Experienced spiderlings preferred chemical cues from the prey in which they were raised. They suffered high mortality on Drosophila flies and attained largest size on the optimal prey. We show here that majority of spiderlings are born with an innate preference to their focal prey, which can be altered by familiarisation with alternative prey, irrespective of whether such a prey is beneficial.
- MeSH
- Stimulation, Chemical MeSH
- Spiders physiology MeSH
- Predatory Behavior physiology MeSH
- Food Preferences physiology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔTm up to 24 °C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.
Human histone deacetylase 6 (HDAC6) is the major deacetylase responsible for removing the acetyl group from Lys40 of α-tubulin (αK40), which is located lumenally in polymerized microtubules. Here, we provide a detailed kinetic analysis of tubulin deacetylation and HDAC6/microtubule interactions using individual purified components. Our data unequivocally show that free tubulin dimers represent the preferred HDAC6 substrate, with a K M value of 0.23 µM and a deacetylation rate over 1,500-fold higher than that of assembled microtubules. We attribute the lower deacetylation rate of microtubules to both longitudinal and lateral lattice interactions within tubulin polymers. Using TIRF microscopy, we directly visualized stochastic binding of HDAC6 to assembled microtubules without any detectable preferential binding to microtubule tips. Likewise, indirect immunofluorescence microscopy revealed that microtubule deacetylation by HDAC6 is carried out stochastically along the whole microtubule length, rather than from the open extremities. Our data thus complement prior studies on tubulin acetylation and further strengthen the rationale for the correlation between tubulin acetylation and microtubule age.
Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.
- MeSH
- Aldehyde Reductase antagonists & inhibitors genetics metabolism MeSH
- Enzyme Inhibitors pharmacology MeSH
- Catalytic Domain genetics MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Humans MeSH
- Models, Molecular MeSH
- Oxidoreductases Acting on Aldehyde or Oxo Group Donors antagonists & inhibitors genetics metabolism MeSH
- Recombinant Proteins genetics metabolism MeSH
- Retinaldehyde metabolism MeSH
- Amino Acid Sequence MeSH
- Structural Homology, Protein MeSH
- Amino Acid Substitution MeSH
- Substrate Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In the last decades, the structural flexibility of cytochromes P450 has been extensively studied by spectroscopic and in silico methods. Here, both approaches are reviewed and compared. Comparison of both methods indicates that the individual cytochromes P450 differ significantly in the flexibilities of their substrate-binding active sites. This finding probably accounts for the large number of isoforms of these enzymes (there are fifty-seven known cytochrome P450 genes in the human genome) and their functional versatility. On the other hand, most of the known cytochrome P450s have a set of common structural features, with an overall structure consisting of a relatively flexible domain (the distal side), a more rigid domain (the heme-binding core) and a domain on the proximal side of the hemoprotein with intermediate flexibility. Substrate access and product egress channels of CYP enzymes are also important structural elements as the majority of these channels are located in the flexible distal side; the location, flexibility, and function of these channels are discussed.
- MeSH
- Protein Conformation MeSH
- Humans MeSH
- Molecular Dynamics Simulation MeSH
- Spectrum Analysis methods MeSH
- Substrate Specificity MeSH
- Cytochrome P-450 Enzyme System chemistry MeSH
- Binding Sites MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75 000 and 525 000 M-1 s-1 for HDAC7 and HDAC5, respectively. Results reported here could help in developing class IIa HDACs inhibitors and also in the search for new natural class IIa HDACs substrates.
Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.
The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.
- MeSH
- Proteasome Inhibitors pharmacology chemistry MeSH
- Catalytic Domain * MeSH
- Proteasome Endopeptidase Complex * metabolism chemistry MeSH
- Protozoan Proteins chemistry metabolism antagonists & inhibitors genetics MeSH
- Substrate Specificity MeSH
- Trichomonas vaginalis * enzymology MeSH
- Publication type
- Journal Article MeSH
Histone deacetylase 6 (HDAC6) is a multidomain cytosolic hydrolase acting mostly on nonhistone protein substrates. Investigations of the substrate specificity of HDAC6 are confounded by the presence of 2 catalytically active deacetylase domains (DD1 and DD2). In this study, acetylome peptide microarrays and peptide libraries were used to map the substrate specificity of DD1 and DD2 of human HDAC6. The results show that DD1 is solely responsible for the deacetylation of substrates harboring the acetyllysine at their C terminus, whereas DD2 exclusively deacetylates peptides with an internal acetyllysine residue. Also, statistical analysis of the deacetylation data revealed amino acid preferences at individual positions flanking the acetyllysine, where glycine and arginine residues are favored at positions N-terminal to the central acetyllysine; negatively charged glutamate is strongly disfavored throughout the sequence. Finally, the deacylation activity of HDAC6 was profiled by using a panel of acyl derivatives of the optimized peptide substrate and showed that HDAC6 acts as a proficient deformylase. Our data thus offer a detailed insight into the substrate preferences of the individual HDAC6 domains at the peptide level, and these findings can in turn help in elucidating the biologic roles of the enzyme and facilitate the development of new domain-specific inhibitors as research tools or therapeutic agents.-Kutil, Z., Skultetyova, L., Rauh, D., Meleshin, M., Snajdr, I., Novakova, Z., Mikesova, J., Pavlicek, J., Hadzima, M., Baranova, P., Havlinova, B., Majer, P., Schutkowski, M., Barinka, C. The unraveling of substrate specificity of histone deacetylase 6 domains using acetylome peptide microarrays and peptide libraries.
- MeSH
- HEK293 Cells MeSH
- Histone Deacetylase 6 chemistry metabolism MeSH
- Catalytic Domain * MeSH
- Humans MeSH
- Lysine chemistry metabolism MeSH
- Peptide Fragments chemistry metabolism MeSH
- Static Electricity MeSH
- Substrate Specificity MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
