Site-directed Mutagenesis Experiments Research Articles

Heterologous production of new lasso peptide koreensin based on genome mining.

Lasso peptides are a class of ribosomally biosynthesized and posttranslationally modified peptides with a knot structure as a common motif. Based on a genome search, a new biosynthetic gene cluster of lasso peptide was found in the genome of the proteobacterium Sphingomonas koreensis. Interestingly, the amino acid sequence of the precursor peptide gene includes two cell adhesion motif sequences (KGD and DGR). Heterologous production of the new lasso peptide was performed using the cryptic biosynthetic gene cluster of S. koreensis. As a result, a new lasso peptide named koreensin was produced by the gene expression system in the host strain Sphingomonas subterranea. The structure of koreensin was determined by NMR and ESI-MS analysis. The three-dimensional structure of koreensin was obtained based on an NOE experiment and the coupling constants. A variant peptide (koreensin-RGD), which had RGD instead of KGD, was produced by heterologous production with site-directed mutagenesis experiment. Koreensin and koreensin-RGD did not show cell adhesion inhibitory activity, although the molecules possessed cell adhesion motifs. The possible presence of a salt bridge between the motifs in koreensin was indicated, and it may prevent the cell adhesion motif from functioning.

Characterization of XtjR8: A novel esterase with phthalate-hydrolyzing activity from a metagenomic library of lotus pond sludge

A fosmid metagenomic library containing 9.7 × 104 clones was constructed. A novel esterase, XtjR8, was isolated through functional screening. XtjR8 shared the maximum amino acid identity (44%) with acetyl-hydrolase from Streptomyces hygroscopicus, and was classified into family IV esterase. XtjR8 exhibited the highest hydrolytic activity for p-nitrophenyl acetate at 40 °C and pH 8.0, and presented more than 40% activity from 20 °C to 80 °C. More importantly, XtjR8 displayed the ability to hydrolyze both phthalate monoesters and diesters, this feature is extremely rare among previously reported esterases. Site-directed mutagenesis experiments revealed that the catalytic triad residues were Ser152, Glu246, and His276. Among them, Ser152 formed a hydrogen bond with dibutyl phthalate (DBP) by molecular docking, Gly84, Gly85, and Leu248 of conserved motifs formed hydrophobic interactions with DBP, respectively, which were important for the catalytic activity. Considering its wide range of temperature and hydrolytic potential toward phthalate esters, XtjR8 will be served as an interesting candidate for biodegradation and industrial applications.

ELA/APELA precursor cleaved by furin displays tumor suppressor function in renal cell carcinoma through mTORC1 activation.

Apelin is a well-established mediator of survival and mitogenic signaling through the apelin receptor (Aplnr) and has been implicated in various cancers; however, little is known regarding Elabela (ELA/APELA) signaling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here, we identified a function of mTORC1 signaling as an essential mediator of ELA that repressed kidney tumor cell growth, migration, and survival. Moreover, sunitinib and ELA showed a synergistic effect in repressing tumor growth and angiogenesis in mice. The use of site-directed mutagenesis and pharmacological experiments provided evidence that the alteration of the cleavage site of proELA by furin induced improved ELA antitumorigenic activity. Finally, a cohort of tumors and public data sets revealed that ELA was only repressed in the main human kidney cancer subtypes, namely clear cell, papillary, and chromophobe renal cell carcinoma. Aplnr was expressed by various kidney cells, whereas ELA was generally expressed by epithelial cells. Collectively, these results showed the tumor-suppressive role of mTORC1 signaling mediated by ELA and established the potential use of ELA or derivatives in kidney cancer treatment.

Biochemical characterization of 2-phosphinomethylmalate synthase from Streptomyces hygroscopicus: A member of the DRE-TIM metallolyase superfamily

2-Phosphinomethylmalate synthase (PMMS) from Streptomyces hygroscopicus catalyzes the first step in the biosynthesis of the herbicide bialophos using 3-phosphinopyruvic acid and acetyl coenzyme A as substrates to form 2-phosphinomethylmalic acid and coenzyme A. PMMS belongs to the Claisen condensation-like (CC-like) subgroup of the DRE-TIM metallolyase superfamily, which uses conserved active site architecture to catalyze a functionally-diverse set of reactions. Analysis of a sequence similarity network for the CC-like subgroup identified PMMS and the related R-citrate synthase in an early-diverging cluster suggesting that this group of sequences are more distinct in relation to other Claisen-condensation subgroup members. To better understand the structure/function landscape of the CC-like subgroup PMMS was recombinantly expressed in Escherichia coli, purified, and characterized with respect to its enzymatic properties. Using oxaloacetate as a substrate analog, the recombinantly-produced enzyme exhibited improved Michaelis constants relative to the previously reported natively-produced enzyme. Results from pH rate profiles and kinetic isotope effects were consistent with results from other members of the CC-like subgroup supporting acid-base chemistry and hydrolysis of the direct Claisen-condensation product as the rate-determining step. Results of site-directed mutagenesis experiments indicate that PMMS uses an active-site architecture similar to homocitrate synthase to select for a dicarboxylic acid substrate.

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton–electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.

Overexpression of FAM46A, a Non-canonical Poly(A) Polymerase, Promotes Hemin-Induced Hemoglobinization in K562 Cells.

FAM46A belongs to the FAM46 subfamily of the nucleotidyltransferase-fold superfamily and is predicted to be a non-canonical poly(A) polymerase. FAM46A has been linked to several human disorders including retinitis pigmentosa, bone abnormality, cancer, and obesity. However, its molecular and functional characteristics are largely unknown. We herein report that FAM46A is expressed in cells of the hematopoietic system and plays a role in hemin-induced hemoglobinization. FAM46A is a nucleocytoplasmic shuttle protein modified by Tyr-phosphorylation only in the cytosol, where it is closely associated with ER. On the other hand, it is located proximal to the chromatin regions of active transcription in the nucleus. FAM46A is a cell cycle-dependent poly-ubiquitinated short-lived protein degraded mostly by proteasome and its overexpression inhibits cell growth and promotes hemin-induced hemoglobinization in K562 cell. Site-directed mutagenesis experiments confirm the non-canonical poly(A) polymerase activity of FAM46A is essential for enhanced hemin-induced hemoglobinization. In summary, FAM46A is a novel poly(A) polymerase that functions as a critical intracellular modulator of hemoglobinization.

Yin Yang 1 Is Essential for Transcriptional Activation of the Bovine Sirt2 Gene in Preadipocytes.

Sirtuin 2 (Sirt2) belongs to the NAD+-dependent deacetylase family, is more highly expressed than other family members in adipocytes, and plays crucial roles in a wide range of biological processes. However, the mechanisms underlying Sirt2 expression during adipogenesis are poorly studied. In this study, the transcriptional start site (TSS) of Sirt2 was identified and two alternative transcript variants were spliced from Sirt2. The 5'-regulatory region of Sirt2 was also characterized; no TATA-box or CCAAT-box was presented in the 5'-flanking region. Two cytosine-phosphate diester-guanine (CpG) islands were also identified between nucleotides -563 and +4. A dual-luciferase reporter assay revealed that a 178 base pair sequence upstream from the TSS (+1) was the core promoter of Sirt2. Results from a site-directed mutagenesis experiment, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay indicated Yin Yang 1 (YY1) to be a positive regulator of bovine Sirt2 in preadipocytes. YY1 is likely to suppress adipogenesis in two different ways by regulating peroxisome proliferator-activated receptor gamma expression. Our results expand the information on the regulatory network of adipogenesis, which is an important basis for improving beef quality, treating obesity, and other related diseases.

Ionic Coulomb blockade and the determinants of selectivity in the NaChBac bacterial sodium channel

Mutation-induced transformations of conductivity and selectivity in NaChBac bacterial channels are studied experimentally and interpreted within the framework of ionic Coulomb blockade (ICB), while also taking account of resonant quantised dehydration (QD) and site protonation. Site-directed mutagenesis and whole-cell patch-clamp experiments are used to investigate how the fixed charge Qf at the selectivity filter (SF) affects both valence selectivity and same-charge selectivity. The new ICB/QD model predicts that increasing ∣Qf∣ should lead to a shift in selectivity sequences toward larger ion sizes, in agreement with the present experiments and with earlier work. Comparison of the model with experimental data leads to the introduction of an effective charge Qf∗ at the SF, which was found to differ between Aspartate and Glutamate charged rings, and also to depend on position within the SF. It is suggested that protonation of the residues within the restricted space of the SF is important in significantly reducing the effective charge of the EEEE ring. Values of Qf∗ derived from experiments on divalent blockade agree well with expectations based on the ICB/QD model and have led to the first demonstration of ICB oscillations in Ca2+ conduction as a function of the fixed charge. Preliminary studies of the dependence of Ca2+ conduction on pH are qualitatively consistent with the predictions of the model.

An Early Association between the α-Helix of the TEAD Binding Domain of YAP and TEAD Drives the Formation of the YAP:TEAD Complex.

The Hippo pathway is an evolutionarily conserved signaling pathway that is involved in the control of organ size and development. The TEAD transcription factors are the most downstream elements of the Hippo pathway, and their transcriptional activity is regulated via the interaction with different co-regulators such as YAP. The structure of the YAP:TEAD complex shows that YAP binds to TEAD via two distinct secondary structure elements, an α-helix and an Ω-loop, and site-directed mutagenesis experiments revealed that the Ω-loop is the "hot spot" of this interaction. While much is known about how YAP and TEAD interact with each other, little is known about the mechanism leading to the formation of a complex between these two proteins. Here we combine site-directed mutagenesis with pre-steady-state kinetic measurements to show that the association between these proteins follows an apparent one-step binding mechanism. Furthermore, linear free energy relationships and a Φ analysis suggest that binding-induced folding of the YAP α-helix to TEAD occurs independently of and before formation of the Ω-loop interface. Thus, the binding-induced folding of YAP appears not to conform to the concomitant formation of tertiary structure (nucleation-condensation) usually observed for coupled binding and folding reactions. Our findings demonstrate how a mechanism reminiscent of the classical framework (diffusion-collision) mechanism of protein folding may operate in disorder-to-order transitions involving intrinsically disordered proteins.

Kohn-Sham Density Functional Calculations Reveal Proton Wires in the Enolization and Carboxylase Reactions Catalyzed by Rubisco.

Ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) plays a fundamental role in the carbon cycle by fixing the atmospheric CO2 used in photosynthesis. Rubisco is all the more remarkable because it must catalyze some difficult multistep reaction chemistry involving proton transfers within the one active site. In the present study, we have used Kohn-Sham density functional theory at the B3LYP/6-31G* level with basis set superposition error and dispersion corrections (B3LYP-gCP-D3) to examine the possibility that the proton transfers can take place through molecular wires (including active-site water molecules) via the classical Grotthuss proton-shuttle mechanism. The results support an essential role for water molecules found in the crystal structures of Rubisco complexes as facilitators of proton transport in all the rate-limiting (catalytic) reaction steps through a network of short proton wires within the Rubisco active site. We suggest that completion of the initial product turnover (cycle) requires two excess protons produced in the initial carbamylation that is required for Rubisco activation. By use of proton wires, a large number of reaction steps may be accommodated within a single active site without necessitating the input of excessive conformational strain energy arising from the movement of residue side chains into positions where direct protonation of substrates can occur. The involvement of the identified types of proton wires in the kinetic mechanism is capable of providing a unique explanation for various experimental observations, including deuterium isotope effects and the results of site-directed mutagenesis experiments, and may thus provide a realistic solution to the problem of Rubisco's challenging chemistry.

Computational-based discovery of FAK FERM domain chemical probes that inhibit HER2-FAK cancer signaling.

The N-terminal FERM domain of focal adhesion kinase (FAK) contributes to FAK scaffolding and interacts with HER2, an oncogene and receptor tyrosine kinase. The interaction between HER2 and FAK drives resistance to FAK-kinase domain inhibitors through FAK Y397 transphosphorylation and FAK re-activation upon inhibition. As such, FAK FERM remains an attractive drug discovery target. In this report, we detail an alternative approach to targeting FAK through virtual screening-based discovery of chemical probes that target FAK FERM. We validated the binding interface between HER2 and FAK using site-directed mutagenesis and GST pull-down experiments. We assessed the ligandability of key-binding residues of HER2 and FAK utilizing computational tools. We developed a virtual screening method to screen ~200,000 compounds against the FAK FERM domain, identifying 20 virtualchemical probes. We performed GST pull-down screening on these compounds, discovering two hits, VS4 and VS14, with nanomolar IC50 s in disrupting HER2-FAK. We performed further testing, including molecular docking, immunofluorescence, phosphorylation, and cellular invasion assays to evaluate the compounds' biological effects. One probe, VS14, was identified with the ability to block both auto- and transphosphorylation of Y397. In all, these studies identify two new probes that target FAK FERM, enabling future investigation of this domain.

Common activation mechanism of class A GPCRs.

Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

Interactions of the effector ExoU from Pseudomonas aeruginosa with short-chain phosphatidylinositides provide insights into ExoU targeting to host membranes

Pseudomonas aeruginosa is an opportunistic multidrug-resistant pathogen and a common cause of infection in cystic fibrosis and ventilator-associated pneumonia and in burn and wound patients. P. aeruginosa uses its type III secretion system to secrete various effector proteins directly into mammalian host cells. ExoU is a potent type III secretion system effector that, after secretion, localizes to the inner cytoplasmic membrane of eukaryotic cells, where it exerts its phospholipase A2 activity upon interacting with ubiquitin and/or ubiquitinated proteins. In this study, we used site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the interaction of ExoU with soluble analogs of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). We found that dioctanoyl PI(4,5)P2 binds to and induces conformational changes in a C-terminal four-helix bundle (4HB) domain of ExoU implicated previously in membrane binding. Other soluble phosphoinositides also interacted with the 4HB but less effectively. Molecular modeling and ligand docking studies indicated the potential for numerous hydrogen bond interactions within and between interhelical loops of the 4HB and suggested several potential interaction sites for PI(4,5)P2 Site-directed mutagenesis experiments confirmed that the side chains of Gln-623 and Arg-661 play important roles in mediating PI(4,5)P2-induced conformational changes in ExoU. These results support a mechanism in which direct interactions with phosphatidylinositol-containing lipids play an essential role in targeting ExoU to host membrane bilayers. Molecules or peptides that block this interaction may prove useful in preventing the cytotoxic effects of ExoU to mitigate the virulence of P. aeruginosa strains that express this potent phospholipase toxin.

Mechanism of Biocatalytic Friedel-Crafts Acylation by Acyltransferase from Pseudomonas protegens.

Acyltransferases isolated from Pseudomonas protegens (PpATase) and Pseudomonas fluorescens (PfATase) have recently been reported to catalyze the Friedel–Crafts acylation, providing a biological version of this classical organic reaction. These enzymes catalyze the cofactor-independent acylation of monoacetylphloroglucinol (MAPG) to diacetylphloroglucinol (DAPG) and phloroglucinol (PG) and have been demonstrated to have a wide substrate scope, making them valuable for potential applications in biocatalysis. Herein, we present a detailed reaction mechanism of PpATase on the basis of quantum chemical calculations, employing a large model of the active site. The proposed mechanism is consistent with available kinetics, mutagenesis, and structural data. The roles of various active site residues are analyzed. Very importantly, the Asp137 residue, located more than 10 Å from the substrate, is predicted to be the proton source for the protonation of the substrate in the rate-determining step. This key prediction is corroborated by site-directed mutagenesis experiments. Based on the current calculations, the regioselectivity of PpATase and its specificity toward non-natural substrates can be rationalized.

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines.

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative 13 protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.

Bioinformatic and biochemical analysis of the key binding sites of the pheromone binding protein of Cyrtotrachelus buqueti Guerin-Meneville (Coleoptera: Curculionidea).

The bamboo snout beetle Cyrtotrachelus buqueti is a widely distributed wood-boring pest found in China, and its larvae cause significant economic losses because this beetle targets a wide range of host plants. A potential pest management measure of this beetle involves regulating olfactory chemoreceptors. In the process of olfactory recognition, pheromone-binding proteins (PBPs) play an important role. Homology modeling and molecular docking were conducted in this study for the interaction between CbuqPBP1 and dibutyl phthalate to better understand the relationship between PBP structures and their ligands. Site-directed mutagenesis and binding experiments were combined to identify the binding sites of CbuqPBP1 and to explore its ligand-binding mechanism. The 3D structural model of CbuqPBP1 has six a-helices. Five of these a-helices adopt an antiparallel arrangement to form an internal ligand-binding pocket. When docking dibutyl phthalate within the active site of CbuqPBP1, a CH-π interaction between the benzene ring of dibutyl phthalate and Phe69 was observed, and a weak hydrogen bond formed between the ester carbonyl oxygen and His53. Thus, Phe69 and His53 are predicted to be important residues of CbuqPBP1 involved in ligand recognition. Site-directed mutagenesis and fluorescence assays with a His53Ala CbuqPBP1 mutant showed no affinity toward ligands. Mutation of Phe69 only affected binding of CbuqPBP1 to cedar camphor. Thus, His53 (Between α2 and α3) of CbuqPBP1 appears to be a key binding site residue, and Phe69 (Located at α3) is a very important binding site for particular ligand interactions.

Molecular and structural basis of nucleoside diphosphate kinase–mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus

The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encoding gene copies as assessed by qPCR. Using gene-knockout and complementation experiments, we found that AfNDK regulates spore and sclerotia development and is involved in plant virulence as assessed in corn and peanut seed-based assays. An antifungal test with the inhibitor azidothymidine suppressed AfNDK activity in vitro and prevented spore production and sclerotia formation in A. flavus, confirming AfNDK's regulatory functions. Crystallographic analysis of AfNDK, coupled with site-directed mutagenesis experiments, revealed three residues (Arg-104, His-117, and Asp-120) as key sites that contribute to spore and sclerotia development. These results not only enrich our knowledge of the regulatory role of this important protein in A. flavus, but also provide insights into the prevention of A. flavus infection in plants and seeds, as well as into the structural features relevant for future antifungal drug development.

Adaptive Changes in Hemoglobin Function in High-Altitude Tibetan Canids Were Derived via Gene Conversion and Introgression.

A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb–O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the β-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked β-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the β-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb–O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed’s relatively short duration of residency at high altitude.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant-negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function.

Crystal Structure and Biochemical Characterization of Ketol-Acid Reductoisomerase from Corynebacterium glutamicum

l-Valine belongs to the branched-chain amino acids (BCAAs) and is an essential amino acid that is crucial for all living organisms. l-Valine is industrially produced by the nonpathogenic bacterium Corynebacterium glutamicum and is synthesized by the BCAA biosynthetic pathway. Ketol-acid reductoisomerase (KARI) is the second enzyme in the BCAA pathway and catalyzes the conversion of (S)-2-acetolactate into (R)-2,3-dihydroxy-isovalerate, or the conversion of (S)-2-aceto-2-hydroxybutyrate into (R)-2,3-dihydroxy-3-methylvalerate. To elucidate the enzymatic properties of KARI from C. glutamicum (CgKARI), we successfully produced CgKARI protein and determined its crystal structure in complex with NADP+ and two Mg2+ ions. Based on the complex structure, docking simulations, and site-directed mutagenesis experiments, we revealed that CgKARI belongs to Class I KARI and identified key residues involved in stabilization of the substrate, metal ions, and cofactor. Furthermore, we confirmed the difference in the binding of metal ions that depended on the conformational change.