Heterodimeric Enzyme Research Articles

Self-Boosting Programmable Release of Multiple Therapeutic Agents by Activatable Heterodimeric Prodrug-Enzyme Assembly for Antitumor Therapy.

Endogenous stimuli-responsive prodrugs, due to their disease lesion specificity and reduced systemic toxicity, have been widely explored for antitumor therapy. However, reactive oxygen species (ROS) as classical endogenous stimuli in the tumor microenvironment (TME) are not enough to achieve the expected drug release. Herein, a ROS-activatable heterodimeric prodrug-loaded enzyme assembly is developed for self-boosting programmable release of multiple therapeutic agents. The heterodimeric prodrug NBS-TK-PTX (namely NTP) is composed of 5-(ethylamino)-9-diethylaminobenzo[a]phenothiazinium chloride analog (NBS), paclitaxel (PTX) and ROS-responsive thioketal (TK) linker, which shows a strong binding affinity with glucose oxidase (GOx), thus obtaining NTP@GOx assembly. Notably, the enzymatic activity of GOx in NTP@GOx is inhibited by NTP. The programmable release is achieved by following steps: i) NTP@GOx is partially dissociated in acidic TME, thus releasing a small segment of NTP and GOx. Thereupon, the enzymatic activity of GOx is recovered; ii) GOx-triggered pH reduction further facilitates the dissociation of NTP@GOx, thus accelerating a large amount of NTP and GOx release; iii) The TK linker of prodrug NTP is cleaved by hydrogen peroxide generated by GOx catalysis, thus expediting the release of NBS for Type-I photodynamic therapy and PTX for chemotherapy, respectively. The NTP@GOx shows great potential for multimodal synergistic cancer therapy.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

Soluble guanylyl cyclase beta1 subunit targets epithelial-to-mesenchymal transition and downregulates Akt pathway in human endometrial and cervical cancer cells

Endometrial and cervical cancer are among the most frequently diagnosed malignancies globally. Nitric oxide receptor-soluble guanylyl cyclase (sGC) is a heterodimeric enzyme composed of two subunits, α1 and β1. Previously we showed that sGCα1 subunit promotes cell survival, proliferation, and migration, but the role of sGCβ1 subunit has not been addressed. The aim of the present work was to study the impact of sGCβ1 restoration in proliferation, survival, migration, and cell signaling in endometrial and cervical cancer cells. We found that sGCβ1 transcript levels are reduced in endometrial and cervical tumors vs normal tissues. We confirmed nuclear enrichment of sGCβ1, unlike sGCα1. Overexpression of sGCβ1 reduced cell viability and augmented apoptotic index. Cell migration and invasion were also negatively affected. All these sGCβ1-driven effects were independent of sGC enzymatic activity. sGCβ1 reduced the expression of epithelial-to-mesenchymal transition factors such as N-cadherin and β-catenin and increased the expression of E-cadherin. sGCβ1 impacted signaling in endometrial and cervical cancer cells through significant downregulation of Akt pathway affecting some of its main targets such as GSK-3β and c-Raf. Our results show for the first time that sGCβ1 exerts several antiproliferative actions in ECC-1 and HeLa cell lines by targeting key regulatory pathways.

ATP1A1-linked diseases require a malfunctioning protein product from one allele

Heterozygous germline variants in ATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αβ) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/− knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/− mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a plasma-membrane protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Paradoxes of Cellular SUMOylation Regulation: A Role of Biomolecular Condensates?

Protein SUMOylation is a major post-translational modification essential for maintaining cellular homeostasis. SUMOylation has long been associated with stress responses as a diverse array of cellular stress signals are known to trigger rapid alternations in global protein SUMOylation. In addition, while there are large families of ubiquitination enzymes, all small ubiquitin-like modifiers (SUMOs) are conjugated by a set of enzymatic machinery comprising one heterodimeric SUMO-activating enzyme, a single SUMO-conjugating enzyme, and a small number of SUMO protein ligases and SUMO-specific proteases. How a few SUMOylation enzymes specifically modify thousands of functional targets in response to diverse cellular stresses remains an enigma. Here we review recent progress toward understanding the mechanisms of SUMO regulation, particularly the potential roles of liquid-liquid phase separation/biomolecular condensates in regulating cellular SUMOylation during cellular stresses. In addition, we discuss the role of protein SUMOylation in pathogenesis and the development of novel therapeutics targeting SUMOylation. SIGNIFICANCE STATEMENT: Protein SUMOylation is one of the most prevalent post-translational modifications and plays a vital role in maintaining cellular homeostasis in response to stresses. Protein SUMOylation has been implicated in human pathogenesis, such as cancer, cardiovascular diseases, neurodegeneration, and infection. After more than a quarter century of extensive research, intriguing enigmas remain regarding the mechanism of cellular SUMOylation regulation and the therapeutic potential of targeting SUMOylation.

The Operon as a Conundrum of Gene Dynamics and Biochemical Constraints: What We Have Learned from Histidine Biosynthesis.

Operons represent one of the leading strategies of gene organization in prokaryotes, having a crucial influence on the regulation of gene expression and on bacterial chromosome organization. However, there is no consensus yet on why, how, and when operons are formed and conserved, and many different theories have been proposed. Histidine biosynthesis is a highly studied metabolic pathway, and many of the models suggested to explain operons origin and evolution can be applied to the histidine pathway, making this route an attractive model for the study of operon evolution. Indeed, the organization of his genes in operons can be due to a progressive clustering of biosynthetic genes during evolution, coupled with a horizontal transfer of these gene clusters. The necessity of physical interactions among the His enzymes could also have had a role in favoring gene closeness, of particular importance in extreme environmental conditions. In addition, the presence in this pathway of paralogous genes, heterodimeric enzymes and complex regulatory networks also support other operon evolution hypotheses. It is possible that histidine biosynthesis, and in general all bacterial operons, may result from a mixture of several models, being shaped by different forces and mechanisms during evolution.

How Single Amino Acid Substitutions Can Disrupt a Protein Hetero-Dimer Interface: Computational and Experimental Studies of the LigAB Dioxygenase from Sphingobium sp. Strain SYK-6.

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity.

Effects of Sodium Dodecyl Sulfate on the Enzyme Catalysis and Conformation of a Recombinant γ-Glutamyltranspeptidase from Bacillus licheniformis.

The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate(SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55°C was greatly reduced from 495.1min to 7.4min in the presence of 1mM SDS. The I3/I1 ratio ofpyreneexcimerfluorescence emission changed around 3.7mMSDSin the absence ofBlrGGT and the inflection point of enzyme samples was reduced to less than 2.7mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0mM. Theintrinsic fluorescence spectraof tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.

Time evolution of the substrate-induced millisecond allosteric activation of imidazole glycerol phosphate synthase.

Deciphering the molecular mechanisms of enzymatic allosteric regulation requires the structural characterization of functional states and also their time evolution toward the formation of the allosterically activated ternary complex. The transient nature and usually slow millisecond time scale interconversion between these functional states hamper their experimental and computational characterization. Here, we design a computational strategy that combines molecular dynamics simulations, enhanced sampling techniques, and dynamical networks to describe the millisecond allosteric activation of imidazole glycerol phosphate synthase (IGPS) from the inactive substrate-free form to the formation of the enzyme active complex. IGPS is a heterodimeric enzyme complex whose HisH subunit is responsible for hydrolysing glutamine and delivering ammonia for the cyclase activity in HisF. Essential molecular details of the long-range millisecond allosteric activation of IGPS remain hidden. Without using a priori information of the active state, our simulations uncover how IGPS, with the allosteric effector bound, spontaneously captures glutamine in a catalytically inactive HisH conformation, subsequently attains a closed HisF:HisH interface, and finally forms the oxyanion hole in HisH for efficient glutamine hydrolysis. We show that the combined effector and substrate binding dramatically decreases the conformational barrier associated with the oxyanion hole formation, in line with experimentally observed 4500-fold activity increase in glutamine hydrolysis. This computational strategy tailored to describe millisecond timescale events can be generalized to study other allosterically regulated enzymes.

Mechanistic insight into impact of phosphorylation on the enzymatic steps of farnesyltransferase.

Farnesyltransferase (FTase) is a heterodimeric enzyme, which catalyzes covalent attachment of the farnesyl group to target proteins, thus coordinating their trafficking in the cell. FTase has been demonstrated to be highly expressed in cancer and neurological diseases; hence considered as a hot target for therapeutic purposes. However, due to the nonspecific inhibition, there has been only one inhibitor that could be translated into the clinic. Importantly, it has been shown that phosphorylation of the α-subunit of FTase increases the activity of the enzyme in certain diseases. As such, understanding the impact of phosphorylation on dynamics of FTase provides a basis for targeting a specific state of the enzyme that emerges under pathological conditions. To this end, we performed 18 μs molecular dynamics (MD) simulations using complexes of (non)-phosphorylated FTase that are representatives of the farnesylation reaction. We demonstrated that phosphorylation modulated the catalytic site by rearranging interactions between farnesyl pyrophosphate (FPP)/peptide substrate, catalytic Zn2+ ion/coordinating residues and hot-spot residues at the interface of the subunits, all of which led to the stabilization of the substrate and facilitation of the release of the product, thus collectively expediting the reaction rate. Importantly, we also identified a likely allosteric pocket on the phosphorylated FTase, which might be used for specific targeting of the enzyme. To the best of our knowledge, this is the first study that systematically examines the impact of phosphorylation on the enzymatic reaction steps, hence opens up new avenues for drug discovery studies that focus on targeting phosphorylated FTase.

A peroxisomal heterodimeric enzyme is involved in benzaldehyde synthesis in plants

Benzaldehyde, the simplest aromatic aldehyde, is one of the most wide-spread volatiles that serves as a pollinator attractant, flavor, and antifungal compound. However, the enzyme responsible for its formation in plants remains unknown. Using a combination of in vivo stable isotope labeling, classical biochemical, proteomics and genetic approaches, we show that in petunia benzaldehyde is synthesized via the β-oxidative pathway in peroxisomes by a heterodimeric enzyme consisting of α and β subunits, which belong to the NAD(P)-binding Rossmann-fold superfamily. Both subunits are alone catalytically inactive but, when mixed in equal amounts, form an active enzyme, which exhibits strict substrate specificity towards benzoyl-CoA and uses NADPH as a cofactor. Alpha subunits can form functional heterodimers with phylogenetically distant β subunits, but not all β subunits partner with α subunits, at least in Arabidopsis. Analysis of spatial, developmental and rhythmic expression of genes encoding α and β subunits revealed that expression of the gene for the α subunit likely plays a key role in regulating benzaldehyde biosynthesis.

Heterologous expression, on-column refolding and characterization of gamma-glutamyl transpeptidase gene from Bacillus altitudinis IHB B1644: A microbial bioresource from Western Himalayas

Several γ-glutamyl compounds produced from gamma-glutamyltranspeptidase (GGT) have alluring features for food, pharmaceutical, and biotechnology applications. GGT from Bacillus altitudinis IHB B1644 was cloned, followed by an expression in pET-47b(+) Escherichia coli BL21(DE3). Recombinant GGT (BaGGT) gene subsisted of 1755 bp encoding protein with 585 amino acids, predicted molecular weight, and theoretical pI of 63.3 kDa, and 4.92, respectively. Ectopic expression resulted in inclusion bodies formation at 37 °C. The protein was solubilized and different strategies like dialysis, rapid dilution, and on-column refolding were tried to get active recombinant protein (BaGGT1). To get protein in soluble fraction, GGT was over expressed at low temperature (20 °C), and IPTG concentration (0.025 mM) (BaGGT2). The heterodimeric enzyme consisted of molecular weight of 40, and 22 kDa for large and small subunits, respectively. The specific activities for BaGGT1 and BaGGT2 were 263.9, and 497.45 U mg−1, respectively. BaGGT1 and BaGGT2 showed optimum temperature, and pH at 37 °C and 9, respectively. Km values of 0.8 and 0.2 mM, and Vmax of 666.67 and 333.33 U mg−1 of protein, were calculated for BaGGT1and BaGGT2, respectively. The results demonstrate potential of BaGGT in biosynthesis of γ-glutamyl compounds with industrial application.

Human immunodeficiency virus type 1 impairs sumoylation.

During infection, the human immunodeficiency virus type 1 (HIV-1) manipulates host cell mechanisms to its advantage, thereby controlling its replication or latency, and evading immune responses. Sumoylation is an essential post-translational modification that controls vital cellular activities including proliferation, stemness, or anti-viral immunity. SUMO peptides oppose pathogen replication and mediate interferon-dependent anti-viral activities. In turn, several viruses and bacteria attack sumoylation to disarm host immune responses. Here, we show that HIV-1 impairs cellular sumoylation and targets the host SUMO E1-activating enzyme. HIV-1 expression in cultured HEK293 cells or in CD4+ Jurkat T lymphocytes diminishes sumoylation by both SUMO paralogs, SUMO1 and SUMO2/3. HIV-1 causes a sharp and specific decline in UBA2 protein levels, a subunit of the heterodimeric SUMO E1 enzyme, which likely serves to reduce the efficiency of global protein sumoylation. Furthermore, HIV-1-infected individuals display a significant reduction in total leukocyte sumoylation that is uncoupled from HIV-induced cytopenia. Because sumoylation is vital for immune function, T-cell expansion and activity, loss of sumoylation during HIV disease may contribute to immune system deterioration in patients.

Genomic identification, annotation, and comparative analysis of Vacuolar-type ATP synthase subunits in Diaphorinacitri.

The hemipteran insect Diaphorina citri, or Asian citrus psyllid, is a vector for Candidatus Liberibacter asiaticus (CLas), the bacterium causing citrus greening disease, or Huanglongbing (HLB). Millions of citrus trees have been destroyed, and every grove in Florida, USA, has been directly affected by this disease. In eukaryotes, vacuolar-type ATP synthase (V-ATPase) is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Fifteen putative V-ATPase genes in the D. citri genome were manually curated. Comparative genomic analysis revealed that D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Annotation of the D. citri genome is a critical step towards developing directed pest management strategies to reduce the spread of HLB throughout the citrus industry.

Mechanistic insight into impact of phosphorylation on dynamics of farnesyltransferase: targeting the protein under hyperinsulinemia

Farnesyltransferase (FTase) is a heterodimeric enzyme that adds the farnesyl group to target proteins such as Ras, thus modulating its function in the cell. The phosphorylation of the α-subunit of FTase has been shown to increase the activity of the protein under hyperinsulinemia, which is prevalent in cancer; however, molecular mechanism has remained elusive. To address this, we performed a total of 18 microseconds molecular dynamics simulations using the systems that are included in the farnesylation process: i)FTase+farnesylpyrophosphate(FPP), ii)FTase+FPP+Kras-peptide(substrate), iii)FTase+farnesylated Kras-peptide(product), which are named as binary, ternary and product, respectively.

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´′- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´′-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.

Human tRNAs with inosine 34 are essential to efficiently translate eukarya-specific low-complexity proteins.

The modification of adenosine to inosine at the wobble position (I34) of tRNA anticodons is an abundant and essential feature of eukaryotic tRNAs. The expansion of inosine-containing tRNAs in eukaryotes followed the transformation of the homodimeric bacterial enzyme TadA, which generates I34 in tRNAArg and tRNALeu, into the heterodimeric eukaryotic enzyme ADAT, which modifies up to eight different tRNAs. The emergence of ADAT and its larger set of substrates, strongly influenced the tRNA composition and codon usage of eukaryotic genomes. However, the selective advantages that drove the expansion of I34-tRNAs remain unknown. Here we investigate the functional relevance of I34-tRNAs in human cells and show that a full complement of these tRNAs is necessary for the translation of low-complexity protein domains enriched in amino acids cognate for I34-tRNAs. The coding sequences for these domains require codons translated by I34-tRNAs, in detriment of synonymous codons that use other tRNAs. I34-tRNA-dependent low-complexity proteins are enriched in functional categories related to cell adhesion, and depletion in I34-tRNAs leads to cellular phenotypes consistent with these roles. We show that the distribution of these low-complexity proteins mirrors the distribution of I34-tRNAs in the phylogenetic tree.

γ-Glutamyltranspeptidase essential for the metabolism of γ-glutamyl compounds in bacteria and its application.

The enzymatic characteristics of γ-glutamyltranspeptidase were elucidated. The catalytic nucleophile of the enzymatic reaction of Escherichia coli γ-glutamyltranspeptidase was identified as the Oγ of the N-terminal Thr-residue of the small subunit. It was demonstrated that the inactive precursor of γ-glutamyltranspeptidase is processed autocatalytically and intramolecularly into the active heterodimeric mature enzyme via an ester intermediate. The catalytic nucleophile of this processing reaction was identified as the same Oγ atom of the N-terminal Thr-residue of the small subunit. These results were also supported by the three-dimensional structures of the γ-glutamyl enzyme intermediate and of the precursor-mimicked T391A nonprocessable mutant enzyme. Applications of transpeptidation and hydrolysis activities of bacterial γ-glutamyltranspeptidases were developed. Using transpeptidation activity, efficient enzymatic production of useful γ-glutamyl compounds, such as prodrug for Parkinson's disease, theanine and kokumi compound, was enabled. Hydrolysis activity was used as glutaminase and the mutant enzymes gaining glutaryl-7-aminocephalosporanic acid acylase activity were isolated.

Functional and structural investigation of N-terminal domain of the SpTad2/3 heterodimeric tRNA deaminase

Editing is a post-transcriptional process that changes the content of nucleic acids occurring on both DNA and RNA levels. Inosine at position 34 in tRNA is one such example, commonly produced via the deamination of A34, catalyzed by adenosine deaminase acting on tRNA (ADAT or Tad). The formation of inosine is essential for cell viability. The eukaryotic deaminases normally consist of the catalytic subunit Tad2 and the structural subunit Tad3, but the catalytic process is poorly understood. Despite the conservation of the (pseudo-) catalytic domains, the heterodimeric enzyme Tad2/3 also possesses additional domains that could exhibit novel functions. Here we present the structure of the N-terminal domain of the Schizosaccharomyces pombe Tad2/3 heterodimeric tRNA(A34) deaminase (N-SpTad2), which shares ~30% sequence identities with uridine-cytidine or pantothenate kinases, but lacks the predicted kinase functions. While biochemical assays indicated that the domain is not a nucleic-acid binder, it is able to significantly influence the A34-tRNA deamination activity of the holoenzyme. Through co-expression and purification analyses, we deduce that N-SpTad2 plays a role in mediating protein-protein contacts and enhancing the stability and solubility of SpTad2/3, without which the deaminase is not functional. Taken together, our structural and biochemical studies highlighted the importance of the additional domains to the intrinsic deaminase functions of heterodimeric Tad2/3 enzymes and promoted our understanding on this essential post-transcriptional tRNA modification.