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Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly- discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

Aminoacyl-tRNA synthetases (AARS) activate amino acids and transfer them to cognate tRNAs. Some AARS cannot achieve the required specificity in the initial amino acid recognition at the synthetic site. Those AARS can incorrectly activate non-cognate amino acids and transfer them to tRNA. To correct the mistake and ensure faithful protein biosynthesis, these AARSs evolved a separate editing domain aimed to hydrolyze erroneously aminoacylated tRNAs (post-transfer editing). To understand what shaped the selectivity of the editing site and how it is related to the initial amino acid selectivity at the synthetic site, we used Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme.1-3 We investigated the synthetic and editing reactions of IleRS using a wide range of amino acids, including non-proteinogenic and synthetic: Val, Nva, Leu, Thr, Met, Ser, Ala, Abu, Nle, F2Abu and F3Abu. Among these, only Val and Nva were poorly discriminated at the synthetic site (i.e. well activated and transferred to the tRNA) and thus can be considered as threat to the fidelity of translation. To our surprise, tRNAs misaminoacylated with all tested amino acids were rapidly hydrolyzed at the editing domain. Thus, it appears that amino acid’s physicochemical features and how well it is rejected at the synthetic site does not pose a significant difference. Only the hydrolysis of cognate Ile-tRNAIle (misediting) was slow, suggesting that the need to keep the cognate product out of editing strongly shaped the specificity of the editing domain. Detailed kinetic analysis revealed that IleRS employs Thr246 and His333 for specific destabilization of Ile-tRNAIle hydrolysis – strategy also known as negative catalysis. Such design enabled IleRS to have broad substrate acceptance at the editing site whilst maintaining a high specificity towards preventing the futile post-transfer editing cycles. This was the first time such broad substrate specificity, paired with negative catalysis, was observed for an AARS, and as such, it marks a new moment in the understanding these vital and ancient enzymes.

Aminoacyl-tRNA synthetases (AARS) activate amino acids and transfer them to cognate tRNAs. Some AARS cannot establish the required specificity in the initial amino acid recognition at the synthetic site and thus may erroneously activate non-cognate amino acids and transfer them onto tRNA. To ensure the faithful protein biosynthesis, those aminoacyl-tRNA synthetases evolved a separate editing domain aimed to hydrolyse erroneously aminoacylated tRNAs (post-transfer editing). To understand what shaped the selectivity of the editing site we used Escherichia coli isoleucyl-tRNA synthetase as a model enzyme. We investigated the kinetics of synthetic and editing reactions of IleRS using a wide range of amino acids, from proteinogenic to synthetic: Val, Nva, Leu, Thr, Met, Ser, Ala, Abu (α-aminobutyrate), Nle (norleucine), F2Abu (γ, γ- difluoro-α-aminobutyrate) and F3Abu (γ, γ, γ- trifluoro-α-aminobutyrate). Among these, only Val and Nva were poorly discriminated (well activated and transferred to the tRNA) and thus can be considered as a threat to the fidelity of translation. To our surprise, tRNAs misaminoacylated with all tested amino acids were rapidly hydrolysed at the editing domain, showing that amino acid’s physicochemical features or how well they are discriminated at the synthetic site and consequently whether or not they can jeopardize the translational fidelity, does not make a significant difference. Only the hydrolysis of cognate Ile-tRNAIle (misediting) was slow, suggesting that the need to keep the cognate product out of editing strongly shaped the specificity of the editing domain. Detailed kinetic analysis revealed that isoleucyl-tRNA synthetase employs Thr246 and His333 for specific destabilization of Ile-tRNAIle hydrolysis (i.e. negative catalysis). Such design enabled isoleucyl-tRNA synthetase to have broad substrate acceptance at the editing site whilst maintaining a high specificity towards preventing the futile post-transfer editing cycles. This was the first time such broad substrate specificity, paired with negative catalysis, was observed for an AARS, and as such, it marks a new moment in the understanding of these vital and ancient enzymes. Živković I., et al. ; Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases ; Nucleic Acids Research, 50 (2022) 4029-4041.

Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl- D-isoglutamine) is the smallest peptidoglycan fragment with adjuvant activity. MDP acts as a pathogen-associated molecular pattern and activates the NOD2 receptor. We have designed and prepared MDP analogs, desmuramyl peptides containing lypophilic 2-aminoadamantane-2- carboxylic acid, and furthermore modified it with mannose moieties. Here we present the design and synthesis of desmuramyl peptides containing 2- aminoadamantane-2-carboxylic acid, and its mono- and di-mannosylated derivatives. They exhibited an improved immunological activity in vivo in the mouse model, and the di-mannoslyated derivative showed to be the most active one. Therefore, molecular-dynamic simulations of NOD2 receptor complexes and manosylated desmuramyl peptides were performed. The intact structure of the NOD2 protein with seven loops, which were omitted in the experimentally solved structure, was modeled and the binding of synthesized mannose mimetics of MDP to NOD2 was investigated. Two potential binding sites have been identified, one located within the NBD domain and the other on the concave portion of the LRR surface of the NOD2 domain.

Muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl- D-isoglutamine) is a conserved fragment of a bacterial cell walls and the smallest peptidoglycan fragment competent to trigger the immune response. MDP binds to NOD2 (nucleotide binding and oligomerization domain-containing protein 2) with affinity in the nanomolar range. Activation of NOD2 by MDP stimulates downstream signaling pathways and promote immune responses. Minor MDP structure modifications affect molecular recognition by NOD2, and thereby influence the modulation of the immune response. Here we present the synthesis of MDP analogs, desmuramyl peptides (DMPs) with incorporated lipophilic adamantly groups at the N-terminus of the dipeptide pharmacophore. The adamantly group was introduced in two ways, via 2-aminoadamantan- 2-carboxylic acid and via (R)-adamantylglycine. Molecular docking simulations of NOD2 receptor and prepared adamantly DMPs were also performed in order to investigate interactions established between DMPs and receptor protein.

Additional file 1. Detailed description of parameterization process.Description of optGM parameterization scheme and preparation of PUB pept dataset.

Additional file 5. Details of GDMIN and optGM comparison.Description of procedure, values of quality metrics and correlation graphs.

Additional file 6. Details of empirical charge method comparison.Description of procedure, values of quality metrics and correlation graphs.

Histamine is a mediator of many different important processes such as contraction of smooth muscle tissues and gastric acid secretion. It is also engaged in neurotransmission and immunomodulation. Because of its important roles, insufficient histamine degradation may cause adverse effects. There are two pathways of histamine degradation, diamine oxidase (DAO) catalyze direct oxidative deamination of histamine to imidazole acetaldehyde.[1, 2] In the other, histamine N-methyltransferase catalyzes the transfer of a methyl group to the secondary amino group of the imidazole ring forming N-methylhistamine. This compound is further metabolized by monoamine oxidase B (MAO B) forming N-methyl-imidazole acetaldehyde. In this work, we combined molecular dynamics simulations, [3] quantum mechanical cluster calculations[3] and EVB simulations to elucidate the catalytic mechanism of MAO B and DAO catalyzed oxidative deamination of N-methylhistamine and histamine. Our results suggest that the in vivo degradation of histamine to yield an imine always occurs via the same catalytic mechanism, namely direct hydride transfer from histamine and N-methylhistamine to the corresponding cofactor (TPQ or FAD, respectively), regardless of the histamine catabolic pathway. [1] T. Bieganski, J. Kusche, K. D. Feussner, R. Hesterberg, H. Richter, W. Lorenz, Arch. Immunol. Ther. Exp. 28, (1980) 901. [2] T. Bieganski, J. Kusche, K. D. Feussner, R. Hesterberg, H. Richter, W. Lorenz, Agents Actions, 10, (1980) 108. [3] A. Marsavelski, R. Vianello, Chem-Eur J. 23, (2017) 2915.

Two highly homologous extracellular enzymes of the SGNH hydrolase supefamily-lipase from Streptomyces rimosus (SrLip)1, 2 and phospholipase A1 from Streptomyces albidoflavus (SaPLA1)3, 4 revealed the catalytic dyad Ser and His instead of common triad Ser-His-Asp. Their different substrate specificity has intrigued us to examine closely characteristic modes of ligand binding into corresponding pockets available in the enzyme interiors, and the influence of the phosphate polar head in the case phospholipid activity. SaPLA1 is preferably phospholipase with a preference towards long acyl chains, showing no detectable lipase activity towards triacylglycerol 1- palmitoyl-2-oleoyl-3-linoleoyl-glycerol (POL). The maximum activity SaPLA1 shows towards 1- palmitoyl-2-oleoyl-glycerol-3-phosphate (POPA) whereas half of the activity is observed with 1-palmitoyl-2-oleoyl-glycerol-3-phosphocholine (POPC).4 On the other hand, SrLip shows both phospholipase and lipase activity, as well as thioesterase, and Tweenase activities, however it prefers shorter acyl chains.1 To understand this substrate promiscuity, we performed docking using both AutoDock Vina and SwissDock to obtain complexes of POPA, POPC, POL and SaPLA. We then performed classical molecular dynamics simulations of apo SaPLA and SaPLA in complex with POPA, POPC and POL to understand the stability and conformational changes in binding. Each structure was simulated in triplicates for 150 ns giving in total 450 ns per structure. Cluster analysis using dihedral angles of amino acids that line the hydrophobic pocket revealed that chi1 dihedral angle of Tyr142 with the average value of -170 degrees is characteristic for productive binding of POPA. The value of -60 degrees is characteristic for apo SaPLA and for non- productive complex with POL. The analysis of the hydrogen bonds has revealed a network that extends from Tyr142 located in the hydrophobic pocket towards the surface of the enzyme. It was shown that when the substrate is productively bound, this network is broken, and Tyr142 does not interact with the side chain of Asp10, but rather forms T-shaped stacking interaction with Phe145. This extensive research requires systematic comparison of ligand binding into interior of these two studied enzymes based on: i) aliphatic chains lengths, ii) saturated vs unsaturated chains and iii) phospholipase vs lipase substrates.

Human heparanase (HPSE) cleaves heparan sulfate (HS) localized in the extracellular matrix allowing immune response to occur as well as cell proliferation and angiogenesis. On the other hand, increased HPSE activity promotes a variety of pathological conditions such as arterial thrombosis and tumor metastasis[1], making HPSE an important drug target. Although crystal structures of HSPE are available[2], the nature of interactions with its very flexible and highly negatively charged polysaccharide substrate HS is not fully understood. To better understand processes that govern substrate recognition, we have explored the ligand binding mechanism of HPSE by analysing its conformational landscape with molecular dynamics (MD) simulations and multi- conformer models generated by crystallographic ensemble refinement. Our results imply that HS binding to HPSE is a complex process. Heparan sulfate is stabilized and immobilized at the active site, whereas the outer regions of the enzyme binding site in complex with HS are characterized with dynamic ionic interactions and exhibit substantial conformational flexibility (Fig. 1). This dynamic substrate binding mechanism allows HSPE to retain high affinity towards its substrate while minimizing the entropic costs associated with binding highly flexible molecules. [3]

Usmeno izlaganje.

U disertaciji je proučen katalitički mehanizam te supstratna specifičnost enzima MAO B i DAO koji su uključeni u katabolizam histamina. Naime, pokazano je kako je izmijenjena aktivnost i ekspresija enzima MAO B povezana s neurodegenerativnim bolestima poput Parkinsonove bolesti te neurološkim poremećajima, poput psihoze, depresije i impulzivnosti. S druge strane, izmijenjena aktivnost enzima DAO razlog je intolerancije na histamin, stanja koje je popraćeno neugodnim simptomima i probavnim problemima. Stoga, svrha ove disertacije je pouzdano definiranje katalitičkog mehanizma oba enzima te utvrđivanje determinanti specifičnosti primjenom najsuvremenijih metoda u računalnoj biokemiji. Rezultati disertacije mogli bi poslužiti kao temelj u racionalnom dizajnu lijekova za liječenje navedenih patoloških stanja. U radu su primijenjene računalne metode koje se koriste za proučavanje strukture, dinamike, selektivnosti i reaktivnosti enzima kao što su molekulsko-dinamičke simulacije, izračun Gibbsove energije vezanja, kvantno-mehanički računi u okviru klaster pristupa te metoda EVB. Molekulsko-dinamičke simulacije spomenutih enzima te izračun Gibbsove energije vezanja rezultirali su objašnjenjem supstratne specifičnosti enzima MAO B, dok su kvantno-mehanički računi u okviru klaster pristupa te metoda EVB dali kvantitativni uvid u katalitički mehanizam oba proučavana enzima. The scope of this dissertation is the elucidation of the catalytic mechanism and substrate specificity of MAO B and DAO enzymes involved in the catabolism of histamine. Altered expression and activity of MAO B is associated with neurodegenerative diseases such as Parkinson's disease, and neurological disorders such as psychosis, depression and impulsivity. On the other hand, the altered enzyme activity of DAO is the root cause for the histamine intolerance, a condition which is characterized by unpleasant symptoms and gastrointestinal problems. Therefore, the purpose of this dissertation is to establish the catalytic mechanism of both enzymes together with the determinants of their specificity using the most advanced methods in computational biochemistry such as molecular dynamics simulations, binding free energy and quantum mechanical calculations within the cluster approach and the empirical valence bond (EVB) simulations. Molecular dynamics simulations and the binding free energy calculations explain the substrate specificity of MAO B enzyme, while the quantum mechanical calculations together with the EVB simulations provide a quantitative insight into the catalytic mechanism of both enzymes.

Histamine plays an important role in the human body and is involved in more than twenty different physiological processes. Due to histamine potent physiological activity, its degradation has to be carefully regulated to avoid adverse reactions. The major routes of histamine inactivation in mammals include monoamine oxidase B (MAO B) and diamine oxidase (DAO) enzymes. The fact that MAO B metabolizes only N-methylhistamine while DAO prefers histamine1-2, pinpoints their remarkable selectivity towards two compounds that differ only in one methyl group. The mechanism of enzyme catalysis and specificity are usually elucidated from the differences in mechanistic aspects of enzymatic reactions for each possible substrate, which provide unambiguous quantitative information about the thermodynamics and the kinetics of reaction pathways. Unfortunately, mechanistic studies are not always experimentally approachable. Therefore, we utilized a combination of molecular dynamics (MD) simulations, MM-PBSA binding free energy calculations and quantum mechanical cluster approach to address substrate specificity and mechanism of MAO B catalysis. We have identified favourable hydrophobic interactions between methyl group of the N-methylhistamine substrate and the hydrophobic side chains of the enzyme binding site that keep substrate anchored and properly oriented for the enzymatic reaction. Since histamine is deprived of methyl group it cannot be properly anchored and rotates within the active site, which results in non-productive orientation for the reaction. Quantum-chemical mechanistic analysis within the cluster model of the enzyme revealed higher activation parameters for histamine relative to its N-methyl counterpart, thus aiding in rationalizing the mentioned selectivity. Inspection of the calculated free-energy profiles (Figure 1.) convincingly shows that MAO B selectivity for the N-methylhistamine over histamine is a result of two synergistic effects: lower activation barrier and more favourable reaction thermodynamics. Moreover, reaction pathway obtained from QM calculations is consistent with recently proposed hydride mechanism of MAO B mechanism of catalysis3-4.

Histamine is an important mediator of many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function. Due to its potent pharmacological activity even at very low concentrations, the degradation of histamine has to be carefully regulated to avoid adverse reactions. Two major routes of histamine inactivation in mammals are: (a) methylation of the imidazole ring, catalyzed by histamine N-methyltransferase (HMT), and (b) oxidative deamination of the primary amino group, catalyzed by diamine DAO oxidase (DAO). HMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the secondary imidazole amino group forming N-methylhistamine, and is a highly specific enzyme that does not show significant methylation of other HMT substrates. N-methylhistamine is not active at the histamine receptor sites, and is further metabolized by monoamine oxidase (MAO), a primary degradation enzyme for a range of biogenic and dietary amines in cells, including amine neurotransmitters in the brain, which is why it has been the central pharmacological target for treating depression and Parkinson’s disease for over 60 years. The notion that histamine is essentially not a substrate for MAO, whereas N-methylhistamine is, raises the question of what is the origin of its unexpected MAO B selectivity towards two very similar compounds, yet completely identical in their reactive ethylamine chain parts.

Histamine is an important mediator of many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function. Due to its potent pharmacological activity even at very low concentrations, the degradation of histamine has to be carefully regulated to avoid adverse reactions, since it can even be toxic when it is present either in excess or in the wrong metabolic context. Two major routes of histamine inactivation in mammals are: (a) methylation of the imidazole ring, catalyzed by histamine N-methyltransferase (HMT), and (b) oxidative deamination of the primary amino group, catalyzed by diamine oxidase (DAO). HMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the secondary imidazole amino group forming Nmethylhistamine, and is a highly specific enzyme that does not show significant methylation of other substrates. N-methylhistamine is not active at the histamine receptor sites, and is further metabolized by monoamine oxidase (MAO), a primary degradation enzyme for a range of biogenic and dietary amines in cells, including amine neurotransmitters in the brain, which is why it has been the central pharmacological target for treating depression and Parkinson’s disease for over 60 years. The notion that histamine is essentially not a substrate for MAO, whereas N-methylhistamine is, poses a very important question: for a promiscuous enzyme such as MAO, what is the origin of its unexpected selectivity towards two very similar compounds, yet completely identical in their reactive ethylamine chain parts.

Substrate selectivity of atypical methanogenic-type Seryl-tRNA synthetases-computational approach

Alosterija je jedna od najvažnijih pojava u regulaciji metaboličke i genetske aktivnosti u organizmu. Identifikacija alosteričkih signalnih puteva pomoću računalnih tehnika predstavlja moćan alat u proučavanju ponašanja proteina uslijed vezanja modulatora što može služiti u otkrivanju novih lijekova.

Tumori nastaju nekontroliranim rastom i diobom stanica koja je posljedica disregulacije kontrolnih mehanizama. Ako tumor ostane lokaliziran, riječ je o benignoj izraslini, no ako se stanica tumora odvoji od primarnog tumora, putovat će krvotokom te se pričvrstiti na drugo mjesto i započeti sekundarni tumor. U tom slučaju radi se o malignom tumoru koji je opasan jer metastazira i to najčešće na dobro prokrvljena mjesta čime remeti njihovo funkcioniranje, ali i ugrožava život. Stoga je potrebno poduzeti neki od koraka: operacija, radioterapija, kemoterapija ili čak kombinacija. Često se primjenjuju i kombinacije antitumorskih lijekova koji imaju različite mehanizme djelovanja ili djeluju na različite mete. Postoji nekoliko vrsta antitumorskih lijekova: lijekovi koji djeluju izravno na nukleinske kiseline, antimetaboliti, hormonske terapije, inhibitori signalnih puteva i enzima, antitijela, konjugati antitijela, genska terapija te fotodinamička terapija.1 U ovom radu posebna pozornost posvećena je monoklonskim antitijelima, imunomodulatorima, inhibitorima kinaza, inhibitorima signalnih puteva i hormonskoj terapiji. Prikazana je njihova struktura (za neke i sinteza), mehanizam djelovanja te je navedeno za što se koriste. Potom su ukratko opisani nedavni napretci u pristupu liječenja raka i aspekti koji se još razvijaju.