Substrate-binding Residues Research Articles

Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce β-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea.

Many methanogenic archaea synthesize β-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical S-adenosylmethionine (SAM) aminomutases involved in the biosynthesis of Nε-acetyl-β-lysine and β-glutamate in Methanococcus maripaludis C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from Escherichia coli, followed by biochemical characterization. In the presence of l-lysine, SAM, and dithionite, this archaeal KAM had a kcat = 14.3 s-1 and a Km = 19.2 mM. The product was shown to be 3(S)-β-lysine, which is like the well-characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)-β-lysine. We further describe the function of MmarC7_1783, a putative radical SAM aminomutase with a ∼160 amino acid extension at its N-terminus. Bioinformatic analysis of the possible substrate-binding residues suggested a function as glutamate 2,3-aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β-glutamate in cell extracts. β-Glutamate has been known to serve as an osmolyte in select methanogens for a long time, but its biosynthetic origin remained unknown until now. Thus, this study defines the biosynthetic routes for β-lysine and β-glutamate in M. maripaludis and expands the importance and diversity of radical SAM enzymes in all domains of life.

Structural Enzymology of the Cysteine Tryptophylquinone (CTQ) Enzyme GoxA

GoxA from Pseudoalteromonas luteoviolaceae belongs to a recently identified family of enzymes (the LodA‐like proteins) that utilize a post‐translationally derived cysteine tryptophylquinone (CTQ) cofactor for catalysis. In contrast to other tryptophylquinone enzymes, GoxA acts as an oxidase rather than a dehydrogenase, using molecular oxygen to catalyze the oxidative deamination of glycine to produce ammonia, glyoxylate and hydrogen peroxide. The crystal structure of this enzyme revealed a homotetramer with a novel alpha helical domain and extensive inter‐subunit interactions, which account for the positive cooperativity observed toward its substrate by steady‐state kinetics. Soaking GoxA crystals in glycine results in a dramatic color change from yellow to blue, indicating the formation of an unusual intermediate at the CTQ site that was also detected in solution. The structure of this intermediate reveals a Schiff‐base formed between the substrate and CTQ that is stabilized by numerous hydrogen bond contacts with residues both in the active site and from a neighboring protomer. The kinetics and structures of active site mutants reveal the roles of specific residues in substrate binding, cooperativity, and catalysis, and allowed for the detection of another intermediate in the GoxA reaction pathway. This combination of kinetics, spectroscopy and X‐ray crystallography has allowed for the construction of a detailed reaction mechanism for this unusual enzyme with high‐resolution structures now available for key intermediates.

Indole‐3‐glycerol phosphate synthase (IGPS) serving as a drug target in M. tuberculosis

Tuberculosis (TB) is a disease that is caused by a bacteria called Mycobacterium tuberculosis and primarily attacks the lungs but can spread to other areas of the body as well. TB can develop resistance to drugs that may be used to cure the disease. Multi‐drug resistant TB, for example, does not respond to the two most powerful anti‐TB drugs, which are isoniazid and rifampicin. For this reason, finding new ways to target TB are highly needed. One possible way to do this is to target the enzymes in the tryptophan biosynthetic pathway of M. tuberculosis. Studies have suggested that the enzyme indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) coded for by the trpC gene could be a target (Shen et al. 2009). The interactions between ligands and MtIGPS were thus investigated by introducing mutations to residues that had been proposed to play a role in catalysis or binding. Specifically, E168 was mutated to Q and K119 was mutated to R. Predictions for these mutations were that K119 is a catalytic acid and that E168 plays a role in substrate binding. We investigated the impact of these mutations on steady state kinetics and rate‐pH profiles to obtain insights into the roles of these two residues in substrate binding and catalysis. These findings will facilitate the design of MtIGPS inhibitor candidates.

The Influence of Identified Key Residues on Catalytic Activity and Substrate Specificity of Taurocyamine Kinases from Arenicola brasiliensis, An Enzyme in the Phosphagen Kinases Family

Tauracyamine kinases (TKs) are part of the phosphagen kinase family which act as an energy buffering mechanism in some invertebrates and unicellular organisms by catalyzing the transfer of phosphate from ATP to guanidino containing compounds, such as arginine, creatine and tauracycamine. TKs utilize tauroayamine as their primary substrate but many TKs exhibit promiscuity in substrate specificity and can also phosprylate glycocyamine or lumbricine. Multiple amino acid residues have been identified as key residues for substrate binding and/or specificity in the phosphagen kinases; specifically, amino acids residue 62‐72 have been identified as key residues within the guanidino substrate (GS) loop that play an important role in substrate binding and preference. Previous work has shown that a single substitution in this loop can ‘flip’ substrate specificity from tauracyamine to glycocyamine although with a significant drop in activity. Using site‐directed mutagenesis we have created additional substitutions in the GS loop to see if they can improve overall activity for the enzyme while still maintaining a preference for glycocyamine. We will be describing our kinetic analysis (KM, kcat, and kcat/KM) of each of the variants and the wildtype TKs to help determine whether the residues mutated are critical in the determining the enzyme’s preference and overall activity with the two substrates, glycocyamine and tauracyamine. This may lead to insight into how different substrate preferences may have evolved.

Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.

Comparative Analysis and Structural Modeling of Elaeis oleifera FAD2, a Fatty Acid Desaturase Involved in Unsaturated Fatty Acid Composition of American Oil Palm.

Simple SummaryPalm oil has become the world’s most important vegetable oil in terms of production quantity, and its overall demand is exponentially growing with the global population. The fatty acid composition and particularly the oleic/linoleic acid ratio are major factors influencing palm oil quality. In this study, we focused on FAD2, a fatty acid desaturase enzyme involved in the desaturation and conversion of oleic acid to linoleic acid in Elaeis oleifera, identified through in silico annotation analysis. Our phylogenetic and comparative studies revealed two SNP markers, SNP278 and SNP851, significantly correlated with the oleic/linoleic acid contents. Our study provides fundamental insights into the mechanism of fatty acids synthesis in oil palm and could support the application of molecular biology techniques to enhance the enzymatic activity and substrate affinity of EoFAD2.American oil palm (Elaeis oleifera) is an important source of dietary oil that could fulfill the increasing worldwide demand for cooking oil. Therefore, improving its production is crucial and could be realized through breeding and genetic engineering approaches aiming to obtain high-yielding varieties with improved oil content and quality. The fatty acid composition and particularly the oleic/linoleic acid ratio are major factors influencing oil quality. Our work focused on a fatty acid desaturase (FAD) enzyme involved in the desaturation and conversion of oleic acid to linoleic acid. Following the in silico identification and annotation of Elaeis oleifera FAD2, its molecular and structural features characterization was performed to better understand the mechanistic bases of its enzymatic activity. EoFAD2 is 1173 nucleotides long and encodes a protein of 390 amino acids that shares similarities with other FADs. Interestingly, the phylogenetic study showed three distinguished groups where EoFAD2 clustered among monocotyledonous taxa. EoFAD2 is a membrane-bound protein with five transmembrane domains presumably located in the endoplasmic reticulum. The homodimer organization model of EoFAD2 enzyme and substrates and respective substrate-binding residues were predicted and described. Moreover, the comparison between 24 FAD2 sequences from different species generated two interesting single-nucleotide polymorphisms (SNPs) associated with the oleic/linoleic acid contents.

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol–converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3′-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3′HE were also prepared from genistein and 3′-hydroxydaidzein (3′HD or 3′-ODI), respectively. In the following efficacy tests, 3′HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERβ, and they did not have significant SERM efficacy except 6HE showing 10% lower β/α ratio response than that of 17β-estradiol. In DPPH radical scavenging assay, 3′HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3′HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116

Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on Thermoanaerobacterium xylanolyticum TxGH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in KM for p-nitrophenyl-β-D-glucoside, and significant decreases in the kcat, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the kcat/KM by over 60,000-fold, while R786 mutants increased both the KM (40-fold) and kcat (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol−1, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Functional and Structural Characterization of Diverse NfsB Chloramphenicol Reductase Enzymes from Human Pathogens.

ABSTRACTPhylogenetically diverse bacteria can carry out chloramphenicol reduction, but only a single enzyme has been described that efficiently catalyzes this reaction, the NfsB nitroreductase from Haemophilus influenzae strain KW20. Here, we tested the hypothesis that some NfsB homologs function as housekeeping enzymes with the potential to become chloramphenicol resistance enzymes. We found that expression of H. influenzae and Neisseria spp. nfsB genes, but not Pasteurella multocida nfsB, allows Escherichia coli to resist chloramphenicol by nitroreduction. Mass spectrometric analysis confirmed that purified H. influenzae and N. meningitides NfsB enzymes reduce chloramphenicol to amino-chloramphenicol, while kinetics analyses supported the hypothesis that chloramphenicol reduction is a secondary activity. We combined these findings with atomic resolution structures of multiple chloramphenicol-reducing NfsB enzymes to identify potential key substrate-binding pocket residues. Our work expands the chloramphenicol reductase family and provides mechanistic insights into how a housekeeping enzyme might confer antibiotic resistance.IMPORTANCE The question of how new enzyme activities evolve is of great biological interest and, in the context of antibiotic resistance, of great medical importance. Here, we have tested the hypothesis that new antibiotic resistance mechanisms may evolve from promiscuous housekeeping enzymes that have antibiotic modification side activities. Previous work identified a Haemophilus influenzae nitroreductase housekeeping enzyme that has the ability to give Escherichia coli resistance to the antibiotic chloramphenicol by nitroreduction. Herein, we extend this work to enzymes from other Haemophilus and Neisseria strains to discover that expression of chloramphenicol reductases is sufficient to confer chloramphenicol resistance to Es. coli, confirming that chloramphenicol reductase activity is widespread across this nitroreductase family. By solving the high-resolution crystal structures of active chloramphenicol reductases, we identified residues important for this activity. Our work supports the hypothesis that housekeeping proteins possessing multiple activities can evolve into antibiotic resistance enzymes.

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-Directed Mutagenesis

AbstractDuring the past three decades many terpene synthases have been characterised from all kingdoms of life. Enzymes of type I, from bacteria, fungi and protists, commonly exhibit several highly conserved motifs and single residues, and the available crystal structures show a shared α-helical fold, while the overall sequence identity is generally low. Several enzymes have been studied by site-directed mutagenesis, giving valuable insights into terpene synthase catalysis and the intriguing mechanisms of terpene synthases. Some mutants are also preparatively useful and give higher yields than the wild type or a different product that is otherwise difficult to access. The accumulated knowledge obtained from these studies is presented and discussed in this review.1 Introduction2 Residues for Substrate Binding and Catalysis3 Residues with Structural Function4 Residues Contouring the Active Site Cavity5 Other Residues6 Conclusions

Unveiling the Effect of Low pH on the SARS-CoV-2 Main Protease by Molecular Dynamics Simulations.

(1) Background: Main Protease (Mpro) is an attractive therapeutic target that acts in the replication and transcription of the SARS-CoV-2 coronavirus. Mpro is rich in residues exposed to protonation/deprotonation changes which could affect its enzymatic function. This work aimed to explore the effect of the protonation/deprotonation states of Mpro at different pHs using computational techniques. (2) Methods: The different distribution charges were obtained in all the evaluated pHs by the Semi-Grand Canonical Monte Carlo (SGCMC) method. A set of Molecular Dynamics (MD) simulations was performed to consider the different protonation/deprotonation during 250 ns, verifying the structural stability of Mpro at different pHs. (3) Results: The present findings demonstrate that active site residues and residues that allow Mpro dimerisation was not affected by pH changes. However, Mpro substrate-binding residues were altered at low pHs, allowing the increased pocket volume. Additionally, the results of the solvent distribution around S, H, N1 and H1 atoms of the catalytic residues Cys145 and His41 showed a low and high-water affinity at acidic pH, respectively. It which could be crucial in the catalytic mechanism of SARS-CoV-2 Mpro at low pHs. Moreover, we analysed the docking interactions of PF-00835231 from Pfizer in the preclinical phase, which shows excellent affinity with the Mpro at different pHs. (4) Conclusion: Overall, these findings indicate that SARS-CoV-2 Mpro is highly stable at acidic pH conditions, and this inhibitor could have a desirable function at this condition.

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).

Looking into a highly thermostable and efficient recombinant manganese-catalase from Geobacillusthermopakistaniensis

Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-IIGt, previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-IIGt, produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-IIGt. Recombinant Cat-IIGt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529μmolmin-1 mg-1. The apparent Km and kcat values were 75mM and 1.5× 104 s-1 subunit-1, respectively. Recombinant Cat-IIGt was highly thermostable with a half-life of 30min at 100°C. The structural attributes of Cat-IIGt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-IIGt a potential candidate for textile and paper processing industries.

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

Functional Classification of Super-Large Families of Enzymes Based on Substrate Binding Pocket Residues for Biocatalysis and Enzyme Engineering Applications.

Large enzyme families such as the groups of zinc-dependent alcohol dehydrogenases (ADHs), long chain alcohol oxidases (AOxs) or amine dehydrogenases (AmDHs) with, sometimes, more than one million sequences in the non-redundant protein database and hundreds of experimentally characterized enzymes are excellent cases for protein engineering efforts aimed at refining and modifying substrate specificity. Yet, the backside of this wealth of information is that it becomes technically difficult to rationally select optimal sequence targets as well as sequence positions for mutagenesis studies. In all three cases, we approach the problem by starting with a group of experimentally well studied family members (including those with available 3D structures) and creating a structure-guided multiple sequence alignment and a modified phylogenetic tree (aka binding site tree) based just on a selection of potential substrate binding residue positions derived from experimental information (not from the full-length sequence alignment). Hereupon, the remaining, mostly uncharacterized enzyme sequences can be mapped; as a trend, sequence grouping in the tree branches follows substrate specificity. We show that this information can be used in the target selection for protein engineering work to narrow down to single suitable sequences and just a few relevant candidate positions for directed evolution towards activity for desired organic compound substrates. We also demonstrate how to find the closest thermophile example in the dataset if the engineering is aimed at achieving most robust enzymes.

Structural and Functional Basis of Potent Inhibition of Leishmanial Leucine Aminopeptidase by Peptidomimetics.

A leucine aminopeptidase primarily hydrolyzes amino acid leucine from the N-terminus end of proteins and is involved in free amino acid regulation, which makes it a potential therapeutic target against neglected tropical diseases including leishmaniasis. We here report the purification and characterization of the leucine aminopeptidase from Leishmania donovani (LdLAP). Using a set of biophysical and biochemical methods, we demonstrate that this enzyme was properly folded after expression in a bacterial system and catalytically active when supplemented with divalent metal cofactors with synthetic fluorogenic peptides. Subsequently, enzymatic inhibition assay denoted that LdLAP activity was inhibited by peptidomimetics, particularly actinonin, which caused potent inhibition and exhibited stronger binding association with the LdLAP. Stronger association of actinonin with the LdLAP was due to a stable complex formation mostly mediated by hydrogen bonding with catalytic and substrate-binding residues in the C-terminal catalytic domain. With molecular dynamics simulation studies, we demonstrate that peptidomimetics retain their topological space in the LdLAP catalytic pocket and form a stable complex. These results expand the current knowledge of aminopeptidase biochemistry and highlight that specific actinonin or peptidomimetic-based inhibitors may emerge as leads to combat leishmaniasis.

Splitting the Functions of a Mitochondrial Iron/Pyrimidine Carrier

The biosynthesis of the iron-cofactors heme and iron-sulfur [Fe-S] clusters takes place in the interior of the mitochondria, but the mechanism of delivery of iron across the impermeable inner mitochondrion membrane is still unclear. In Saccharomyces cerevisiae the mitochondrial carrier proteins Mrs3 and Mrs4, are involved in iron transport, as is the human homolog mitoferrin 1, but yeast lacking these genes still have normal mitochondrial iron content, suggesting involvement of other proteins. A synthetic lethal screen identified a requirement of Rim2 in the absence of Mrs3 and Mrs4. Rim2 of S. cerevisiae, and the human homologs SLC25A33 and SLC25A36, import and export pyrimidine nucleotides in and out of mitochondria. Rim2 also delivers iron into intact mitochondria, allowing conversion of a fluorescent porphyrin precursor into non-fluorescent heme inside the organelle. Measurement of [Fe-S] assembly, using 35S-cysteine, and loading into the aconitase, demonstrated that Rim2 mediates mitochondrial iron uptake for [Fe-S] cluster biogenesis.Point mutations were generated in the predicted substrate binding site of Rim2. K299 on helix 5 and E248 on helix 4 showed asymmetry and were predicted to be substrate binding residues, and these were mutated respectively to alanine. In intact mitochondria, incorporation of radiolabeled iron (55Fe) into heme, was significantly decreased in the Rim2-E248A mutant. Additionally protein levels of aconitase in the Rim2-E248 mutant were much lower, suggestive of a destabilization of aconitase in the absence of [Fe-S] cluster incorporation. Interestingly, TTP (pyrimidine) homoexchange was entirely preserved in this mutant. The TTP/TMP heteroexchange was compromised, perhaps because the E248 residue was required for proton coupled transport. Conversely for the Rim2-K299A mutant, all pyrimidine exchange functions were severely compromised almost to the point of the Rim2 null. By contrast, iron dependent functions, such as 55Fe transport and incorporation into heme, were maintained at wild-type levels in the K299A mutant.In summary the lysine at position 299 of Rim2 was critical for pyrimidine transport/exchange where as the glutamic acid at position 248 was required for iron transport/use and proton coupled TTP/TMP heteroexchange across the mitochondrial inner membrane. Iron transport by Rim2 is likely to be proton coupled and pyrimidine independent. DisclosuresNo relevant conflicts of interest to declare.

Key amino acid residues in homoserine-acetyltransferase from M. tuberculosis give insight into the evolution of MetX family of enzymes – HAT, SAT and HST

Multiple sequence alignment of homoserine-acetyltransferases, serine-acetyltransferases and homoserine-succinyltransferases show they all belong to MetX family, having evolved from a common ancestor by conserving the catalytic site and substrate binding residues. The discrimination in the substrate selection arises due to the presence of substrate-specific residues lining the substrate-binding pocket. Mutation of Ala59 and Gly62 to Gly and Pro respectively in homoserine-acetyltransferase from M. tuberculosis resulted in a serine-acetyltransferase like enzyme as it acetylated both l-homoserine and l-serine. Homoserine-acetyltransferase from M. tuberculosis when mutated at positon 322 where Leu was converted to Arg, resulted in succinylation over acetylation of l-homoserine. Our studies establish the importance of the substrate binding residues in determining the type of activity possessed by MetX family, despite all of them having the same catalytic triad Ser-Asp-His. Hence key residues at the substrate binding pocket dictate whether the given enzyme shows predominant transferase or hydrolase activity.

Cloning, sequence analysis, and tissue expression of marmoset paraoxonase 1

Paraoxonase (PON) plays roles in the metabolism of organophosphate xenobiotics and drugs. Despite the importance of marmosets for research into drug metabolism and pharmacokinetics, marmoset paraoxonase has not yet been fully characterized. Consequently, we identified the PON1 gene in the marmoset genome by sequence homology analysis. Marmoset PON1 cDNA containing an open reading frame (1065 bp) was successfully cloned from marmoset liver by reverse transcription-polymerase chain reaction. The deduced amino acid sequence (355 amino acids) has approximately 93% identity with the human ortholog and contains important amino acid residues for substrate binding and calcium ion coordination. According to a phylogenetic tree of PON1 amino acid sequences constructed using data from seven animal species, marmoset PON1 is closer to human PON1 than it is to the PON1 orthologs of experimental animals such as pigs, rabbits, rats, and mice. Marmoset PON1 mRNA was predominantly expressed in liver among the five tissues examined. Marmoset PON1 protein secreted into plasma was detected by immunoblotting. The paraoxon-hydrolyzing activity in plasma was higher in marmosets than in humans. Based on these data, we concluded that marmoset and human PON1 have similar characteristics with regard to genomic structure, amino acid sequences, and tissue distribution.