ABSTRACT
Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential (R)-selective amine dehydrogenases, as native amine dehydrogenases are mostly (S)-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features.
Subject(s)
Amines , Amines/metabolism , Amines/chemistry , Substrate Specificity , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Computational Biology/methods , Biocatalysis , Biodiversity , Models, MolecularABSTRACT
The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.
Subject(s)
Catalytic Domain , Oxidoreductases Acting on CH-NH Group Donors , Peptide Initiation Factors , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Crystallography, X-Ray , Eukaryotic Translation Initiation Factor 5A , Lysine/chemistry , Lysine/metabolism , Lysine/analogs & derivatives , Models, Molecular , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Protein BindingABSTRACT
Plant-derived insecticide-neonicotinoid insecticides (NIs) played a crucial role in the development of agriculture and food industry in recent years. Nevertheless, synthesis of these nitrogen-containing heterocyclic compounds with an effective and greener routing remains challenging especially to the notion raise of "green chemistry" and "atom economy". While bio-catalyzed methods mediated by nicotinate dehydrogenase (NDHase) then provide an alternative. The current review mainly focuses on the introduction of sources, components, structure, catalytic mechanism and applications of NDHase. Specifically, NDHase is known as nicotinic acid hydroxylase and the sources principally derived from phylum Proteobacteria. In addition, NDHase requires the participation of the electron respiratory chain system on the cell membrane. And the most important components of the electron respiratory chain are hydrogen carrier, which is mainly composed of iron-sulfur proteins (Fe-S), flavin dehydrogenase (FAD), molybdenum binding protein and cytochromes. Heterologous expression studies were hampered by the plasmid and host with high efficiency and currently only Pseudomonas entomophila L48 as well as Comamonas testosterone was successfully utilized for the expression of NDHase. Furthermore, it is speculated that the conjugate and inductive effects of the substituent group at position 3 of the substrate pyridine ring exerts a critical role in the hydroxylation reactions at position 6 concerning about the substrate molecular recognition mechanism. Finally, applications of NDHase are addressed in terms of pesticide industry and wastewater treatment. On conclusion, this critical review would not only deepen our understanding of the theory about NDHase, but also provides the guideline for future investigation of NDHase.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , PlasmidsABSTRACT
Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.
Subject(s)
Neurodegenerative Diseases , Neurodevelopmental Disorders , Oxidoreductases Acting on CH-NH Group Donors , Peptide Initiation Factors , Humans , Cryoelectron Microscopy , Peptide Initiation Factors/chemistry , Protein Processing, Post-Translational , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Eukaryotic Translation Initiation Factor 5AABSTRACT
Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors , Animals , Catalytic Domain , Humans , Mice , Oxidoreductases/metabolism , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Substrate Specificity , Polyamine OxidaseABSTRACT
Putative tumor suppressor ALDH1L1, the product of natural fusion of three unrelated genes, regulates folate metabolism by catalyzing NADP+-dependent conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Cryo-EM structures of tetrameric rat ALDH1L1 revealed the architecture and functional domain interactions of this complex enzyme. Highly mobile N-terminal domains, which remove formyl from 10-formyltetrahydrofolate, undergo multiple transient inter-domain interactions. The C-terminal aldehyde dehydrogenase domains, which convert formyl to CO2, form unusually large interfaces with the intermediate domains, homologs of acyl/peptidyl carrier proteins (A/PCPs), which transfer the formyl group between the catalytic domains. The 4'-phosphopantetheine arm of the intermediate domain is fully extended and reaches deep into the catalytic pocket of the C-terminal domain. Remarkably, the tetrameric state of ALDH1L1 is indispensable for catalysis because the intermediate domain transfers formyl between the catalytic domains of different protomers. These findings emphasize the versatility of A/PCPs in complex, highly dynamic enzymatic systems.
Subject(s)
Genes, Tumor Suppressor , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Tumor Suppressor Proteins/genetics , Animals , Catalytic Domain , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Rats , Tumor Suppressor Proteins/metabolismABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors , Pseudomonas aeruginosa , Heme/metabolism , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Spermidine , Spermine , Polyamine OxidaseABSTRACT
The metabolic inflammation (meta-inflammation) of obesity is characterized by proinflammatory macrophage infiltration into adipose tissue. Catalysis by deoxyhypusine synthase (DHPS) modifies the translation factor eIF5A to generate a hypusine (Hyp) residue. Hypusinated eIF5A (eIF5AHyp) controls the translation of mRNAs involved in inflammation, but its role in meta-inflammation has not been elucidated. Levels of eIF5AHyp were found to be increased in adipose tissue macrophages from obese mice and in murine macrophages activated to a proinflammatory M1-like state. Global proteomics and transcriptomics revealed that DHPS deficiency in macrophages altered the abundance of proteins involved in NF-κB signaling, likely through translational control of their respective mRNAs. DHPS deficiency in myeloid cells of obese mice suppressed M1 macrophage accumulation in adipose tissue and improved glucose tolerance. These findings indicate that DHPS promotes the post-transcriptional regulation of a subset of mRNAs governing inflammation and chemotaxis in macrophages and contributes to a proinflammatory M1-like phenotype.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors , Adipose Tissue/metabolism , Animals , Inflammation/metabolism , Macrophages/metabolism , Mice , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , PhenotypeABSTRACT
Based on the novel allosteric site of deoxyhypusine synthase (DHPS), two series of 30 novel 5-(2-methoxyphenoxy)-2-phenylpyrimidin-4-amine derivatives as DHPS inhibitors were designed and synthesized. Among them, compound 8m, with the best DHPS inhibitory potency (IC50 = 0.014 µM), exhibited excellent inhibition against melanoma cells, which was superior to that of GC7. Besides, molecular docking and molecular dynamics (MD) simulations further proved that compound 8m was tightly bound to the allosteric site of DHPS. Flow cytometric analysis and enzyme-linked immunosorbent assay (ELISA) showed that compound 8m could inhibit the intracellular reactive oxygen species (ROS) level. Furthermore, by western blot analysis, compound 8m effectively activated caspase 3 and decreased the expressions of GP-100, tyrosinase, eIF5A2, MMP2, and MMP9. Moreover, both Transwell analysis and wound healing analysis showed that compound 8m could inhibit the invasion and migration of melanoma cells. In the in vivo study, the tumor xenograft model showed that compound 8m effectively inhibited melanoma development with low toxicity.
Subject(s)
Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Melanoma/drug therapy , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Pyrimidines/therapeutic use , Allosteric Site , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Female , Humans , Male , Mice, Inbred BALB C , Mice, Nude , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Protein Binding , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Pyrimidines/pharmacokinetics , Rats, Sprague-Dawley , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least 8 genes including those encoding 3 nonidentical subunits and 3 modifying enzymes. Our heterologous expression study has revealed that the 8 genes are necessary and sufficient for the QHNDH biogenesis.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/biosynthesis , Electrophoresis, Polyacrylamide Gel , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
Dihydromethanopterin reductase (DmrB), is a naturally occurring cage protein found in various archaeal and a few bacterial species. It exists as 24mer with cubic geometry where 8 trimeric subunits are present at the corners of each cube. Each trimer is made up of three monomeric units and six FMN, where two molecules of FMN are present at the interface of each monomer. DmrB is involved in the conversion of dihydromethanopterin to tetrahydromethanopterin using FMN as a redox equivalent. In the present study, we have used spectroscopic and biochemical techniques along with complementary bio-informatic work to understand the assembly principles of the DmrB. Our results show a concentration dependant self-assembly of DmrB which is mediated by ionic interactions. The co-factor FMN stabilizes and preserves the secondary and quaternary structure of DmrB against thermal insult, indicating that the higher order assembly of DmrB is very thermostable. Our work provides an interesting piece of information regarding the role of the co-factors in the thermostability of these classes of cage proteins. The understanding of the assembly and disassembly of this thermostable cage would enable the downstream usage of this system in various nano-biotechnological applications.
Subject(s)
Bacterial Proteins/chemistry , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Protein Multimerization , Pterins/chemistry , Bacterial Proteins/metabolism , Enzyme Stability , Osmolar ConcentrationABSTRACT
Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300-400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.
Subject(s)
Catalase/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Arabidopsis/enzymology , Arabidopsis Proteins/chemistry , Chromatography, Gel/methods , Crystallization , Glutamate Dehydrogenase/chemistry , Molecular Weight , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Protein Structure, Quaternary , Recombinant Proteins/chemistry , X-Ray DiffractionABSTRACT
Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD+, suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q.
Subject(s)
Bacterial Proteins , Nicotine/chemistry , Oxidoreductases Acting on CH-NH Group Donors , Pseudomonas/enzymology , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Oxidoreductases Acting on CH-NH Group Donors/biosynthesis , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/isolation & purification , Pseudomonas/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Deoxyhypusine synthase (DHS) catalyzes the first step of the post-translational modification of eukaryotic translation factor 5A (eIF5A), which is the only known protein containing the amino acid hypusine. Both proteins are essential for eukaryotic cell viability, and DHS has been suggested as a good candidate target for small molecule-based therapies against eukaryotic pathogens. In this work, we focused on the DHS enzymes from Brugia malayi and Leishmania major, the causative agents of lymphatic filariasis and cutaneous leishmaniasis, respectively. To enable B. malayi (Bm)DHS for future target-based drug discovery programs, we determined its crystal structure bound to cofactor NAD+. We also reported an in vitro biochemical assay for this enzyme that is amenable to a high-throughput screening format. The L. major genome encodes two DHS paralogs, and attempts to produce them recombinantly in bacterial cells were not successful. Nevertheless, we showed that ectopic expression of both LmDHS paralogs can rescue yeast cells lacking the endogenous DHS-encoding gene (dys1). Thus, functionally complemented dys1Δ yeast mutants can be used to screen for new inhibitors of the L. major enzyme. We used the known human DHS inhibitor GC7 to validate both in vitro and yeast-based DHS assays. Our results show that BmDHS is a homotetrameric enzyme that shares many features with its human homologue, whereas LmDHS paralogs are likely to form a heterotetrameric complex and have a distinct regulatory mechanism. We expect our work to facilitate the identification and development of new DHS inhibitors that can be used to validate these enzymes as vulnerable targets for therapeutic interventions against B. malayi and L. major infections.
Subject(s)
Anthelmintics/pharmacology , Antiprotozoal Agents/pharmacology , Brugia malayi/drug effects , Enzyme Inhibitors/pharmacology , Helminth Proteins/antagonists & inhibitors , Leishmania major/drug effects , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Amino Acid Sequence , Animals , Anthelmintics/chemistry , Antiprotozoal Agents/chemistry , Brugia malayi/enzymology , Brugia malayi/genetics , Brugia malayi/growth & development , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Helminth Proteins/chemistry , Helminth Proteins/genetics , Helminth Proteins/metabolism , High-Throughput Screening Assays , Leishmania major/enzymology , Leishmania major/genetics , Leishmania major/growth & development , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Sequence AlignmentABSTRACT
Amine oxidases (AOs) including copper containing amine oxidases (CuAOs) and FAD-dependent polyamine oxidases (PAOs) are associated with polyamine catabolism in the peroxisome, apoplast and cytoplasm and play an essential role in growth and developmental processes and response to biotic and abiotic stresses. Here, we identified PAO genes in common wheat (Triticum aestivum), T. urartu and Aegilops tauschii and reported the genome organization, evolutionary features and expression profiles of the wheat PAO genes (TaPAO). Expression analysis using publicly available RNASeq data showed that TaPAO genes are expressed redundantly in various tissues and developmental stages. A large percentage of TaPAOs respond significantly to abiotic stresses, especially temperature (i.e. heat and cold stress). Some TaPAOs were also involved in response to other stresses such as powdery mildew, stripe rust and Fusarium infection. Overall, TaPAOs may have various functions in stress tolerances responses, and play vital roles in different tissues and developmental stages. Our results provided a reference for further functional investigation of TaPAO proteins.
Subject(s)
Cold-Shock Response/genetics , Oxidoreductases Acting on CH-NH Group Donors/genetics , Plant Proteins/genetics , Thermotolerance/genetics , Triticum/genetics , Aegilops/enzymology , Aegilops/genetics , Alternative Splicing , Amino Acid Sequence , Datasets as Topic , Evolution, Molecular , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Genome, Plant , Genome-Wide Association Study , Markov Chains , Models, Genetic , Molecular Weight , Multigene Family , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Phylogeny , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Domains/genetics , RNA-Seq , Sequence Alignment , Triticum/enzymology , Polyamine OxidaseABSTRACT
The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288-P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.
Subject(s)
Oxidoreductases Acting on CH-NH Group Donors/ultrastructure , Peptide Initiation Factors/ultrastructure , Protein Processing, Post-Translational/genetics , Pyrococcus horikoshii/ultrastructure , Binding Sites/genetics , Crystallography, X-Ray , Eukaryota/genetics , Eukaryota/metabolism , Lysine/analogs & derivatives , Lysine/chemistry , Lysine/genetics , Lysine/metabolism , NAD/chemistry , NAD/genetics , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Protein Conformation , Pyrococcus horikoshii/enzymology , Spermidine/chemistry , Spermidine/metabolismABSTRACT
Nicotinate dehydrogenase (NDHase) is a membrane protein with three subunits (ndhS, ndhL, and ndhM), which is difficult to express in a functional form using common hosts such as Escherichia coli, Bacillus subtilis, or Pichia pastoris. Comamonas testosteroni is a suitable microbial chassis for expressing multi-subunit membrane proteins. However, the expression of NDHase in C. testosteroni is extremely low. We have developed a systematic approach to create an efficient protein expression system in C. testosteroni CNB-2 using multi-level N-terminal engineering. We selected a strong promoter for the Mmp1 system that enables control of transcriptional strength in unconventional bacteria. This enhanced the expression of a green fluorescent reporter protein threefold. Following modification of the N-terminal Shine-Dalgarno sequence and rearrangement of amino acid sequence in the starting area of the gene encoding NDHase, enzyme activity increased from 90.6 to 165 U/L. These optimized N-terminal Shine-Dalgarno and amino acid sequences were used to enhance the expression of ndhL subunit and improve the balance expression of three subunits of NDHase, resulting in enzyme activity of 192 U/L that far surpasses the previously reported level. These results highlight a promising strategy for the development of other heterologous expression systems for challenging proteins using unconventional bacteria.
Subject(s)
Comamonas testosteroni/genetics , Genetic Engineering , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/genetics , Genes, Reporter/genetics , Promoter Regions, Genetic/geneticsABSTRACT
Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved "ball-and-chain" motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.
Subject(s)
Lysine/analogs & derivatives , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Humans , Lysine/chemistry , Lysine/metabolism , Models, Molecular , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Protein Binding , Protein ConformationABSTRACT
Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.
Subject(s)
Enzyme Inhibitors/chemistry , Indoles/chemistry , Oxidoreductases Acting on CH-NH Group Donors/antagonists & inhibitors , Thiophenes/chemistry , Allosteric Site , Crystallography, X-Ray , Drug Discovery , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Guanine/analogs & derivatives , Guanine/metabolism , High-Throughput Screening Assays , Humans , Indoles/chemical synthesis , Indoles/metabolism , Molecular Structure , NAD/metabolism , Oxidoreductases Acting on CH-NH Group Donors/chemistry , Oxidoreductases Acting on CH-NH Group Donors/metabolism , Protein Binding , Protein Conformation/drug effects , Spermidine/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/metabolismABSTRACT
Nicotinate dehydrogenase (NDHase) from Comamonas testosteroni JA1 catalyzes the C6â¯hydroxylation of 3-cyanopyridine with high regional selectivity, which is a very difficult and complex reaction for chemical synthesis. However, because NDHase is a membrane protein with three subunits (ndhS, ndhL and ndhM), it is difficult to express the enzyme in a functional form using common hosts such as Escherichia coli, Bacilus subtilis or Pichia pastoris. Furthermore, the enzyme requires special electron transfer chains in the membrane system for proper catalytic activity. Thus, we investigated the expression of NDHase in non-model bacterial strains, which are evolutionarily similar to C. testosteroni JA1, using several broad-host plasmids with different copy numbers as expression vectors. We successfully expressed NDHase in soluble from using the pVLT33 vector in C. testosteroni CNB-2, and found the activity of enzyme to be 40.6 U/L. To further improve the expression of NDHase in C. testosteroni CNB-2, we trialed a T7-like MmP1 system, composed of MmP1 RNA polymerase and an MmP1 promoter, which is used for transcriptional control in non-model bacteria. This increased protein expression and enzyme activity doubled to 90.5 U/L. A molecular chaperone was co-expressed using pBBR1â¯MCS-5 in the same host to improve the efficiency of folding and assembly of multi-subunit structures. The maximum activity was 115 U/L using the molecular chaperone GroES-EL, far surpassing the previously reported level, although expression was almost equivalent. These results indicate that a strategy involving the construction of a T7-like system and co-expression of a molecular chaperone offers an efficient approach for heterologous expression of enzymes that are difficult to express in functional forms using conventional hosts.