Structural engineering and truncation of α-amylase from the hyperthermophilic archaeon Methanocaldococcus jannaschii.

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (µmol min-1 mg -1) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes.

Cryo-EM structure of a 16.5-kDa small heat-shock protein from Methanocaldococcus jannaschii.

The small heat-shock protein (sHSP) from the archaea Methanocaldococcus jannaschii, MjsHSP16.5, functions as a broad substrate ATP-independent holding chaperone protecting misfolded proteins from aggregation under stress conditions. This protein is the first sHSP characterized by X-ray crystallography, thereby contributing significantly to our understanding of sHSPs. However, despite numerous studies assessing its functions and structures, the precise arrangement of the N-terminal domains (NTDs) within this sHSP cage remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of MjsHSP16.5 at 2.49-Å resolution. The subunits of MjsHSP16.5 in the cryo-EM structure exhibit lesser compaction compared to their counterparts in the crystal structure. This structural feature holds particular significance in relation to the biophysical properties of MjsHSP16.5, suggesting a close resemblance to this sHSP native state. Additionally, our cryo-EM structure unveils the density of residues 24-33 within the NTD of MjsHSP16.5, a feature that typically remains invisible in the majority of its crystal structures. Notably, these residues show a propensity to adopt a ß-strand conformation and engage in antiparallel interactions with strand ß1, both intra- and inter-subunit modes. These structural insights are corroborated by structural predictions, disulfide bond cross-linking studies of Cys-substitution mutants, and protein disaggregation assays. A comprehensive understanding of the structural features of MjsHSP16.5 expectedly holds the potential to inspire a wide range of interdisciplinary applications, owing to the renowned versatility of this sHSP as a nanoscale protein platform.

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids.

In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8-4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15-150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins.

Crystal structure of Methanococcus jannaschii dihydroorotase.

In this paper, we report the structural analysis of dihydroorotase (DHOase) from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii. DHOase catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of de novo pyrimidine biosynthesis. DHOases form a very diverse family of enzymes and have been classified into types and subtypes with structural similarities and differences among them. This is the first archaeal DHOase studied by x-ray diffraction. Its structure and comparison with known representatives of the other subtypes help define the structural features of the archaeal subtype. The M. jannaschii DHOase is found here to have traits from all subtypes. Contrary to expectations, it has a carboxylated lysine bridging the two Zn ions in the active site, and a long catalytic loop. It is a monomeric protein with a large ß sandwich domain adjacent to the TIM barrel. Loop 5 is similar to bacterial type III and the C-terminal extension is long.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

A Genetic Study of Nif-Associated Genes in a Hyperthermophilic Methanogen.

Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH, nifD, nifK, and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN, essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCEMethanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

Hyperthermophilic methanogenic archaea act as high-pressure CH4 cell factories.

Bioprocesses converting carbon dioxide with molecular hydrogen to methane (CH4) are currently being developed to enable a transition to a renewable energy production system. In this study, we present a comprehensive physiological and biotechnological examination of 80 methanogenic archaea (methanogens) quantifying growth and CH4 production kinetics at hyperbaric pressures up to 50 bar with regard to media, macro-, and micro-nutrient supply, specific genomic features, and cell envelope architecture. Our analysis aimed to systematically prioritize high-pressure and high-performance methanogens. We found that the hyperthermophilic methanococci Methanotorris igneus and Methanocaldococcoccus jannaschii are high-pressure CH4 cell factories. Furthermore, our analysis revealed that high-performance methanogens are covered with an S-layer, and that they harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase. Thus, high-pressure biological CH4 production in pure culture could provide a purposeful route for the transition to a carbon-neutral bioenergy sector.

Efficient Incorporation of Clickable Unnatural Amino Acids Enables Rapid and Biocompatible Labeling of Proteins in Vitro and in Bacteria.

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment - typically at a single site or at a few sites per protein - of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l-phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production - using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain - of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.

Structural and Functional Insights into an Archaeal Lipid Synthase.

The UbiA superfamily of intramembrane prenyltransferases catalyzes an isoprenyl transfer reaction in the biosynthesis of lipophilic compounds involved in cellular physiological processes. Digeranylgeranylglyceryl phosphate (DGGGP) synthase (DGGGPase) generates unique membrane core lipids for the formation of the ether bond between the glycerol moiety and the alkyl chains in archaea and has been confirmed to be a member of the UbiA superfamily. Here, the crystal structure is reported to exhibit nine transmembrane helices along with a large lateral opening covered by a cytosolic cap domain and a unique substrate-binding central cavity. Notably, the lipid-bound states of this enzyme demonstrate that the putative substrate-binding pocket is occupied by the lipidic molecules used for crystallization, indicating the binding mode of hydrophobic substrates. Collectively, these structural and functional studies provide not only an understanding of lipid biosynthesis by substrate-specific lipid-modifying enzymes but also insights into the mechanisms of lipid membrane remodeling and adaptation.

Structure-affinity insights into the Na+ and Ca2+ interactions with multiple sites of a sodium-calcium exchanger.

Selective recognition and transport of Na+ and Ca2+ ions by sodium-calcium exchanger (NCX) proteins is a primary prerequisite for Ca2+ signaling and homeostasis. Twelve ion-coordinating residues are highly conserved among NCXs, and distinct NCX orthologs contain two or three carboxylates, while sharing a common ion-exchange stoichiometry (3Na+ :1Ca2+ ). How these structural differences affect the ion-binding affinity, selectivity, and transport rates remains unclear. Here, the mutational effects of three carboxylates (E54, E213, and D240) were analyzed on the ion-exchange rates in the archaeal NCX from Methanococcus jannaschii and ion-induced structure-affinity changes were monitored by attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR). The D240N mutation elevated the ion-transport rates by twofold to threefold, meaning that the deprotonation of D240 is not essential for transport catalysis. In contrast, mutating E54 or E213 to A, D, N, or Q dramatically decreased the ion-transport rates. ATR-FTIR revealed high- and low-affinity binding of Na+ or Ca2+ with E54 and E213, but not with D240. These findings reveal distinct structure-affinity states at specific ion-binding sites in the inward-facing (IF) and outward-facing orientation. Collectively, two multidentate carboxylate counterparts (E54 and E213) play a critical role in determining the ion coordination/transport in prokaryotic and eukaryotic NCXs, whereas the ortholog substitutions in prokaryotes (aspartate) and eukaryotes (asparagine) at the 240 position affect the ion-transport rates differently (kcat ), probably due to the structural differences in the transition state.

Role of disordered regions in transferring tyrosine to its cognate tRNA.

Aminoacyl tRNA synthetase (AARS) plays an important role in transferring each amino acid to its cognate tRNA. Specifically, tyrosyl tRNA synthetase (TyrRS) is involved in various functions including protection from DNA damage due to oxidative stress, protein synthesis and cell signaling and can be an attractive target for controlling the pathogens by early inhibition of translation. TyrRS has two disordered regions, which lack a stable 3D structure in solution, and are involved in tRNA synthetase catalysis and stability. One of the disordered regions undergoes disorder-to-order transition (DOT) upon complex formation with tRNA whereas the other remains disordered (DR). In this work, we have explored the importance of these disordered regions using molecular dynamics simulations of both free and RNA-complexed states. We observed that the DOT and DR regions of the first subunit acts as a flap and interact with the acceptor arm of the tRNA. The DOT-DR flap closes when tyrosine (TyrRSTyr) is present at the active site of the complex and opens in the presence of tyrosine monophosphate (TyrRSYMP). The DOT and DR regions of the second subunit interact with the anticodon stem as well as D-loop of the tRNA, which might be involved in stabilizing the complex. The anticodon loop of the tRNA binds to the structured region present in the C-terminal of the protein, which is observed to be flexible during simulations. Detailed energy calculations also show that TyrRSTyr complex has stronger binding energy between tRNA and protein compared to TyrRSYMP; on the contrary, the anticodon is strongly bound in TyrRSYMP. The results obtained in the present study provide additional insights for understanding catalysis and the involvement of disordered regions in Tyr transfer to cognate tRNA.

Cation permeability in CorA family of proteins.

CorA proteins belong to 2-TM-GxN family of membrane proteins, and play a major role in Mg2+ transport in prokaryotes and eukaryotic mitochondria. The selection of substrate is believed to occur via the signature motif GxN, however there is no consensus how strict this selection within the family. To answer this question, we employed fluorescence-based transport assays on three different family members, namely CorA from bacterium Thermotoga maritima, CorA from the archeon Methanocaldococcus jannaschii and ZntB from bacterium Escherichia coli, reconstituted into proteoliposomes. Our results show that all three proteins readily transport Mg2+, Co2+, Ni2+ and Zn2+, but not Al3+. Despite the similarity in cation specificity, ZntB differs from the CorA proteins, as in the former transport is stimulated by a proton gradient, but in the latter by the membrane potential, confirming the hypothesis that CorA and ZntB proteins diverged to different transport mechanisms within the same protein scaffold.

A photo-cross-linking approach to monitor protein dynamics in living cells.

BACKGROUND: Proteins, which comprise one of the major classes of biomolecules that constitute a cell, interact with other cellular factors during both their biogenesis and functional states. Studying not only static but also transient interactions of proteins is important to understand their physiological roles and regulation mechanisms. However, only a limited number of methods are available to analyze the dynamic behaviors of proteins at the molecular level in a living cell. The site-directed in vivo photo-cross-linking approach is an elegant technique to capture protein interactions with high spatial resolution in a living cell. SCOPE OF REVIEW: Here, we review the in vivo photo-cross-linking approach including its recent applications and the potential problems to be considered. We also introduce a new in vivo photo-cross-linking-based technique (PiXie) to study protein dynamics with high spatiotemporal resolution. MAJOR CONCLUSIONS: In vivo photo-cross-linking enables us to capture weak/transient protein interactions with high spatial resolution, and allows for identification of interacting factors. Moreover, the PiXie approach can be used to monitor rapid folding/assembly processes of proteins in living cells. GENERAL SIGNIFICANCE: In vivo photo-cross-linking is a simple method that has been used to analyze the dynamic interactions of many cellular proteins. Originally developed in Escherichia coli, this system has been extended to studies in various organisms, making it a fundamental technique for investigating dynamic protein interactions in many cellular processes. This article is part of a Special issue entitled "Novel major techniques for visualizing 'live' protein molecules" edited by Dr. Daisuke Kohda.

Cryo-electron microscopy structure of an archaeal ribonuclease P holoenzyme.

Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5' maturation. Here we report the cryo-EM structures of Methanocaldococcus jannaschii (Mja) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of MjaRNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3'-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by MjaRNase P. The overall organization of MjaRNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.

The Elongator subunit Elp3 is a non-canonical tRNA acetyltransferase.

The Elongator complex catalyzes posttranscriptional tRNA modifications by attaching carboxy-methyl (cm5) moieties to uridine bases located in the wobble position. The catalytic subunit Elp3 is highly conserved and harbors two individual subdomains, a radical S-adenosyl methionine (rSAM) and a lysine acetyltransferase (KAT) domain. The details of its modification reaction cycle and particularly the substrate specificity of its KAT domain remain elusive. Here, we present the co-crystal structure of bacterial Elp3 (DmcElp3) bound to an acetyl-CoA analog and compare it to the structure of a monomeric archaeal Elp3 from Methanocaldococcus infernus (MinElp3). Furthermore, we identify crucial active site residues, confirm the importance of the extended N-terminus for substrate recognition and uncover the specific induction of acetyl-CoA hydrolysis by different tRNA species. In summary, our results establish the clinically relevant Elongator subunit as a non-canonical acetyltransferase and genuine tRNA modification enzyme.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

Engineering bioorthogonal protein-polymer hybrid hydrogel as a functional protein immobilization platform.

We demonstrate the synthesis of protein-polymer hybrid hydrogel that can be used as a platform for immobilizing functional proteins. Orthogonal chemistry was employed for cross-linking the hybrid network and conjugating proteins to the gel backbone, allowing for the convenient, one-pot formation of a functionalized hydrogel. The resulting hydrogel had tunable mechanical properties, was stable in solution, and biocompatible.

TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1.

Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens.

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.