Aminoacyl Residues Research Articles

RNA-directed peptide synthesis across a nicked loop.

Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N-protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N-acyl-aminoacyl transfer is not stereoselective.

Polymyxin B nonapeptide potentiates the eradication of Gram-negative bacterial persisters.

The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.

Enhanced Molecular Networking Shows Microbacterium sp. V1 as a Factory of Antioxidant Proline-Rich Peptides

Two linear proline-rich peptides (1-2), bearing an N-terminal pyroglutamate, were isolated from the marine bacterium Microbacterium sp. V1, associated with the marine sponge Petrosia ficiformis, collected in the volcanic CO2 vents in Ischia Island (South Italy). Peptide production was triggered at low temperature following the one strain many compounds (OSMAC) method. Both peptides were detected together with other peptides (3-8) via an integrated, untargeted MS/MS-based molecular networking and cheminformatic approach. The planar structure of the peptides was determined by extensive 1D and 2D NMR and HR-MS analysis, and the stereochemistry of the aminoacyl residues was inferred by Marfey's analysis. Peptides 1-8 are likely to arise from Microbacterium V1 tailor-made proteolysis of tryptone. Peptides 1 and 2 were shown to display antioxidant properties in the ferric-reducing antioxidant power (FRAP) assay.

LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain

BackgroundAminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region.ResultsUsing genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human β-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria.ConclusionsOverall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Identification, Characterization, and Synthesis of Natural Parasitic Cysteine Protease Inhibitors: Pentacitidins Are More Potent Falcitidin Analogues.

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogues of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization, and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogues by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogues followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and, additionally, low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

Undescribed Anti-Inflammatory Thalysiaketides from Marine Sponge Clathria (Thalysias) vulpina (Lamarck, 1814).

Two undescribed polyketide-type compounds, thalysiaketide A and thalysiaketide B were isolated from a sponge of marine origin Clathria (Thalysias) vulpina (Lamarck, 1814). Thalysiaketide A exhibited significantly greater inhibitory potential against inflammatory 5-lipoxygenase (IC50 0.87 mM) and cyclooxygense-2 (IC50 0.93 mM) compared to those displayed by its thalysiaketide B analog (IC50 ≥1.05 mM). The 5-lipoxygenase inhibitory activity of thalysiaketide A was considerably superior to ibuprofen (standard, IC50 >4 mM). Higher degree of polar belongings (topological polar surface area 93.06) in conjunction with relatively lower docking parameters of thalysiaketide A with the aminoacyl residues of cyclooxygense-2 and 5-lipoxygenase (docking score -12.99 and -12.27 kcal/mol, respectively) recognized its prospective anti-inflammatory potential.

Novel Furanyl-Substituted Isochromanyl Class of Anti-Inflammatory Turbinochromanone from Brown Seaweed Turbinaria conoides.

Organic extract of the brown seaweed Turbinaria conoides (Sargassaceae) was chromatographically fractionated to yield an undescribed furanyl-substituted isochromanyl metabolite, named as turbinochromanone, which was characterized as methyl 4-[(3S)-8-{[(3R)-4-ethyl-2,3-dihydrofuran-3-yl]methyl}-1-oxo-3,4-dihydro-1H-2-benzopyran-3-yl]butanoate. The isochromanyl derivative possessed comparable attenuation potential against 5-lipoxygenase (IC50 3.70 μM) with standard 5-lipoxygenase inhibitor drug zileuton (IC50 2.41 μM). Noticeably, the index of anti-inflammatory selectivity of turbinochromanone (∼1.7) was considerably greater than that exhibited by the standard agent diclofenac (1.06). Antioxidant properties of turbinochromanone against oxidants (IC50 ∼24 μM) further supported its potential anti-inflammatory property. Greater electronic properties (topological polar surface area of 61.8) along with comparatively lesser docking parameters of the studied compound with aminoacyl residues of targeted enzymes (cyclooxygenase-2 and 5-lipoxygenase) (binding energy of -11.05 and -9.40 kcal mol-1 , respectively) recognized its prospective anti-inflammatory potential. In an aim to develop seaweed-based natural anti-inflammatory leads, the present study isolated turbinochromanone as promising 5-lipoxygenase and cyclooxygenase-2 inhibitor, which could be used for pharmaceutical and biotechnological applications.

Cistobislactone, an undescribed variant of 14-membered bislactonic macrodiolide, from old-lady octopus Cistopus indicus (family Octopodidae) attenuates inflammatory lipoxygenase

Chemical evaluation of specialised metabolites from the old-lady marine octopus Cistopus indicus (family Octopodidae) led to the isolation of an undescribed 14-membered bislactonic macrodiolide cistobislactone, which was characterized as 12-(4'-ethyl-6'-methoxy-3'-methyl-hex-1-enyl)-5,11-dihydroxy-6-methyl-1,7-dioxacyclotetradeca-3,9-diene-2,8-dione. Cistobislactone exhibited noticeably greater inhibitory potential against 5-lipoxygenase (IC50 2.06 mM) compared to standard anti-inflammatory agent ibuprofen (IC50 4.61 mM, p < 0.05). Superior antioxidant properties of cistobislactone against the oxidants (IC50 ∼1.8 mM) also reinforced its promising anti-inflammatory activity. Higher electronic properties (topological polar surface area of 102.3) and balanced hydrophobicity (logarithm of octanol-water coefficient ∼3) could recognize its higher interaction at the enzyme active site resulting in an effective attenuation of 5-lipoxygenase and efficient inter-membrane permeability. Comparatively lesser binding energy (-6.5 kcal mol−1) and docking score (-7.5 kcal mol−1) of cistobislactone with the aminoacyl residues of 5-lipoxygenase could further recognize its anti-inflammatory potential.

Clathriolide from marine demosponge Clathria (Thalysias) vulpina (Lamarck, 1814): previously undescribed macrocylic lactone with attenuating potential against angiotensin converting enzyme

Angiotensin-I converting enzyme catalyses the rate-determined step of the conversion of angiotensin-I to angiotensin-II that narrows the blood vessels, and angiotensin-I converting enzyme inhibitors were recognised as important medications for hypertension-related diseases. Chemical investigation of angiotensin-I converting enzyme inhibitors from marine demospongiae Clathria (Thalysias) vulpina (family Microcionidae), resulted in a previously undescribed 22-membered macrocyclic lactone derivative, named as clathriolide. The studied compound showed potential angiotensin converting enzyme attenuation property (IC50 0.41 mM), which was comparable with the standard captopril (IC50 0.36 mM). Clathriolide revealed significantly greater antioxidant potentials against free radical species (IC50 < 1 mM) in comparison with the standard α-tocopherol (IC50 > 1.5 mM). Superior electronic characteristics (topological polar surface area > 100) coupled with relatively smaller binding energy and docking score of clathriolide with the aminoacyl residues of angiotensin-I converting enzyme (–11.5 and –12.2 kcal/mol, respectively) described its potential inhibitory property against angiotensin-I converting enzyme.

Procerolides A-B from Microcionidae marine sponge Clathria procera: Anti-inflammatory macrocylic lactones with selective cyclooxygenase-2 attenuation properties

Cyclooxygenase-2 has been recognized to catalyze the formation of inflammatory prostaglandins from arachidonic acid. Attenuation potential against cyclooxygenase-2 coupled with greater anti-inflammatory selectivity index were contemplated to be vital indicators to assess anti-inflammatory activities of bioactive compounds. In the present study, two undescribed fourteen-membered macrocyclic lactones, procerolide A and B were isolated to homogeneity from the organic extract of the marine sponge Clathria procera (family: Microcionidae). Procerolide B exhibited greater attenuation potential against cyclooxygenase-2 (IC50 0.89 mM) than that displayed by procerolide A, whereas 5-lipoxygenase inhibitory activity of procerolide B (IC50 1.08 mM) was significantly greater than that displayed by procerolide A (IC50 0.95 mM) and anti-inflammatory agent ibuprofen (IC50 4.50 mM). Additionally, greater anti-inflammatory selectivity index of the procerolides (~1.3) than the synthetic agent (0.43) was accounted for the selective inhibition of the compounds towards cyclooxygenase-2. Higher electronic properties (topological polar surface area of > 100) along with lesser steric properties (molar volume < 300 cm3) of the compounds compared to the standard supported their significant anti-inflammatory potential. Additionally, procerolide B exhibited comparatively lesser binding energy with aminoacyl residues of cyclooxygenase-2 (-9.82 kcal/mol) thereby recognizing its prospective therapeutic use against inflammatory pathogenesis.

Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida.

Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L−1 h−1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L−1 h−1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L−1 after 60 h of fed-batch bioreactor cultivation could be attained.

Kaempferol as a Potential PAK4 Inhibitor in Triple Negative Breast Cancer: Extra Precision Glide Docking and Free Energy Calculation

P-21 activating kinase 4 (PAK4) is implicated in poor prognosis of many human tumors, particularly in Triple Negative Breast Cancer (TNBC) progression. Studies have revealed the crucial role of PAK4 in cell proliferation, anchorage-independent growth and cell migration among other hallmarks of cancer. Thus, PAK4 is an attractive target for anti-TNBC drug design and development. In our research, we used in silico methods to investigate the inhibitory potentials of kaempferol against PAK4 as compared with co-crystallized 4T6 and a standard PAK4 inhibitor-KPT-9274. The ligands were docked into the ATP-binding site of the target enzyme and post-docking validations were calculated. In the molecular docking results, kaempferol had higher affinity than the standard KPT-9274. However, the SP and XP docking scores for the co-crystallized 4T6 were the highest. The analyses of the docking showed a favorable interaction between kaempferol and the catalytic-important aminoacyl residues, especially GLU396, LEU398 and ASP458 in the ATP-binding site of PAK4 when compared with what was obtained in the 4T6-PAK4 complex. Molecular mechanics based MM-GBSA was used to validate docking results. The free energy calculations revealed that kaempferol may have a favorable biological activity. Furthermore, the druggability of each ligand was assessed using the QikProp module and the SwissADME online tool. Kaempferol possessed a propitious drug-like property when compared to the standard ligands. We, therefore, put forward a logical argument that kaempferol can be further evaluated as a potential PAK4 inhibitor in TNBC.

N-Aminoacyl and N-hydroxyacyl derivatives of diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside: Synthesis, antimicrobial and hemolytic activities

Diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside is a semisynthetic saponin with antimicrobial and antitumor activities. To search for more effective analogues, N-aminoacyl and N-hydroxyacyl derivatives of this saponin were synthesized conventionally and with microwave assistance, and tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. None of the tested compounds exhibit activity against Gram-negative bacteria. Almost all of the synthesized N-aminoacyl saponins exhibit antifungal activity and act effectively against Gram-positive bacteria, some better than the parent compound. The best acting saponins are the same size and possess sarcosine or l- or d-alanine attached to the parent glucosaminoside. Shorter and longer aminoacyl residues are less advantageous. d-Alanine derivative is the most effective against Gram positive bacteria. Structure-activity relationship (SAR) analysis indicates that the free α-amino group in aminoacyl residue is necessary for antimicrobial activities of the tested saponins. (N-Acetyl)aminoacyl and N-hydroxyacyl analogs are inactive. Measurements of the hemolytic activities demonstrate that the best acting saponins are not toxic towards human red blood cells.

Inhibition of α-amylases by pentagalloyl glucose: Kinetics, molecular dynamics and consequences for starch absorption

Pentagalloyl-glucose (PGG) occurs in many plants and possesses several therapeutic properties, including anti-diabetic ones. Based on the latter, the inhibitory action of PGG on α-amylases was investigated. PGG inhibited both the pancreatic and the salivary α-amylases. With starch as the substrate, complexation with the free enzyme form either predominates (pancreatic enzyme) or is the only complexation that is effectively allowed (salivary enzyme; competitive inhibition). This contrasts with a previous report on the salivary enzyme showing non-competitive inhibition when a synthetic substrate was used. PGG was less effective than acarbose as an inhibitor of α-amylases and presented a different binding pattern in terms of the aminoacyl residues involved. Finally, PGG also diminished the hyperglycemic excursion normally caused by starch administration when it was given to rats simultaneously to the polysaccharide. This suggests that PGG or foods and infusions rich in PGG could be helpful as adjuvants for maintaining normal glycemia.

A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6–10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.

Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

BackgroundThe gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces.ResultsCelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498–507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans.ConclusionThis study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC.

SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the “Helical Scaffold” Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.

A chemical and computational approach to comprehensive glycation characterization on antibodies

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS2 mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS1 based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.

The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23–25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23–25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.

Cyclolization of D-Lysergic Acid Alkaloid Peptides

The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²⁺/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human.