Structure-function analysis of T4 RNA ligase 2

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.     

Methanogenesis and ATP synthesis in a protoplast system of Methanobacterium thermoautotrophicum.

When Methanobacterium thermoautotrophicum cells were incubated in 50 mM potassium phosphate buffer (pH 7.0) containing 1 M sucrose and autolysate from Methanobacterium wolfei, they were transformed into protoplasts. The protoplasts, which possessed no cell wall, lysed in buffer without sucrose. Unlike whole cells, the protoplasts did not show convoluted internal membrane structures. The protoplasts produced methane from H2-CO2 (approximately 1 mumol min-1 mg of protein-1) at about 50% the rate obtained for whole cells, and methanogenesis was coupled with ATP synthesis. Addition of the protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF-6847) to protoplast suspensions resulted in a dissipation of the membrane potential (delta psi), and this was accompanied by a parallel decrease in the rates of ATP synthesis and methanogenesis. In this respect protoplasts differed from whole cells in which ATP synthesis and methanogenesis were virtually unaffected by the addition of the protonophore. It is concluded that the insensitivity of whole cells to protonophores could be due to internal membrane structures. Membrane preparations produced from lysis of protoplasts or by sonication of whole cells gave comparatively low rates of methanogenesis (methylcoenzyme M methylreductase activity, less than or equal to 100 nmol of CH4 min-1 mg of protein-1), and no coupling with ATP synthesis could be demonstrated.     

Thermodynamic analysis of growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum, an anaerobic archaebacterium using methanogenesis as the catabolic pathway, is characterized by large heat production rates, up to 13 W g⁻¹, and low biomass yields, in the order of 0.02 C‐mol mol⁻¹ H₂ consumed. These values, indicating a possibly “inefficient” growth mechanism, warrant a thermodynamic analysis to obtain a better understanding of the growth process. The growth‐associated heat production (Δ_rH) and the growth‐associated Gibbs energy dissipation per mol biomass formed (Δ_rG) were −3730 kJ C‐mol⁻¹ and −802 kJ C‐mol⁻¹, respectively. The Gibbs energy change found in this study is indeed unusually high as compared to aerobic methylotrophes, but not untypical for methanogens grown on CO₂. It explains the low biomass yield. Based on the information available on the energetic metabolism and on an ATP balance, the biomass yield can be predicted to be approximately in the range of the experimentally determined value. The fact that the exothermicity exceeds vastly even the Gibbs energy change can be explained by a dramatic entropy decrease of the catabolic reaction. Microbial growth characterized by entropy reduction and correspondingly by unusually large heat production may be called entropy‐retarded growth. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 74–81, 1999.     

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid Mth ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K_m 1.1 µM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD⁺ cannot. Mth ligase activity is thermophilic in vitro, with optimal nick-joining at 60°C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase–adenylate intermediate (step 1) and hence cannot seal a 3′-OH/5′-PO₄ nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase–adenylate formation, but defective in activating the nick via formation of the DNA–adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA–adenylate, could be elicited for the wild-type Mth ligase by inclusion of calcium as the divalent cation cofactor. Mth ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that Mth ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

An improved assay method for a pseudomurein-degrading enzyme of Methanobacterium wolfei and the Protoplast formation of Methanobacterium thermoautotrophicum by the enzyme

An improved assay method of a pseudomurein-degrading enzyme and its properties are described. The pseudomurein-degrading enzyme purified from Methanobacterium wolfei autolysate under an anoxic condition was assayed with the cell wall of Methanobacterium thermoautotrophicum as a substrate. By this improved method the enzyme activity was measured quantitatively and reproducibly. Moreover, the cell wall substrate can be stored in a freezer and used as needed, and the time required for an assay was as short as 1 h. The optimum pH and temperature of the enzyme was pH 6.8-7.4 and 75°C, respectively. Although the enzyme lost 50% of the activity upon heating at 75°C for 10 min in the absence of the cell wall substrate, it was more stable against heat inactivation in the presence of the substrate. Furthermore the inactivated enzyme recovered some of the activity by incubating with the substrate. Although the enzyme lost most of the activity under aerobic conditions, the activity was recovered under reducing conditions with Na₂S·9H₂O or DTT (dithiothreitol). The enzyme was also purified under aerobic conditions retaining the same specific activity as the anoxically purified enzyme. Using the partially purified enzyme the conditions preparing protoplasts of M. thermoautotrophicum was established.     

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60–64°C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.     

Structure-function analysis of yeast tRNA ligase

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.     

ATP synthesis driven by a potassium diffusion potential in Methanobacterium thermoautotrophicum is stimulated by sodium

Abstract ATP synthesis driven by a potassium diffusion potential was studied in cell suspensions of Methanobacterium thermoautotrophicum (Marburg). This transient increase in the intracellular ATP content was stimulated five-fold by the addition of sodium ions, from about 2 nmol ATP/min × mg cells (dry weight) at 0.07 mM Na⁺ to about 10 nmol ATP/min × mg cells at 25 mM Na⁺.     

Structure-function correlations derived from faster variants of a RNA ligase deoxyribozyme

We previously reported the in vitro selection of several Mg²⁺-dependent deoxyribozymes (DNA enzymes) that synthesize a 2′–5′ RNA linkage from a 2′,3′-cyclic phosphate and a 5′-hydroxyl. Here we subjected the 9A2 deoxyribozyme to re-selection for improved ligation rate. We found two new DNA enzymes (7Z81 and 7Z48) that contain the catalytic core of 7Q10, a previously reported small deoxyribozyme that is unrelated in sequence to 9A2. A third new DNA enzyme (7Z101) is unrelated to either 7Q10 or 9A2. The new 7Z81 and 7Z48 DNA enzymes have ligation rates over an order of magnitude higher than that of 7Q10 itself and they have additional sequence elements that correlate with these faster rates. Our findings provide insight into structure–function relationships of catalytic nucleic acids.     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.     

Geranylgeranylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.     

ATP activation and properties of the methyl coenzyme M reductase system in Methanobacterium thermoautotrophicum.

The requirement of ATP for the methyl coenzyme M methylreductase in extracts of Methanobacterium thermoautotrophicum was found to be catalytic; for each mol of ATP added, 15 mol of methane was produced from methyl coenzyme M [2-(methylthio)ethanesulfonic acid]. Other nucleotide triphosphates partially replaced ATP in activation of the reductase. All components of the reaction were found in the supernatant fraction of cell extracts after centrifugation at 100,000 X g for 1 h; optimal reaction rates occurred at 65 degrees C, at a pH range of 5.6 to 6.0, and at concentrations of ATP and MgCl2 of 1 mM and 40 mM, respectively. Chloral hydrate, chloroform, nitrite, 2,4-dinitrophenol, and viologen dyes (compounds known to inhibit methanogenesis from a variety of substrates) were found to inhibit the conversion of methyl coenzyme M to methane. Methyl coenzyme M methylreductase was shown to be present in a variety of methanogens.     

Structure-function analysis of yeast RNA debranching enzyme (Dbr1), a manganese-dependent phosphodiesterase

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2′-5′ phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Δdbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure–activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.     

Isolation of a nucleotide activated disaccharide pentapeptide precursor from Methanobacterium thermoautotrophicum

The yeasts Pachysolen tannophilus and Pichia stipitis differed in their ability to utilize D-xylose in the presence of D-fructose. When P. tannophilus was grown aerobically in fructose-xylose mixture, the ketohexose was utilized preferentially over the pentose. However, in P. stipitis cultures, the converse was observed. The effect was associated with the ability of D-fructose to repress the induction of xylose reductase and xylitol dehydrogenase activities in P. tannophilus but not in P. stipitis. Both yeasts grew on D-fructose and fermented it to ethanol when it was supplied as the sole carbon source. The results suggest that there may exist some fundamental difference in the regulation of D-fructose metabolism between P. tannophilus and P. stipitis.     

Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum

A gene encoding superoxide dismutase (SOD) was cloned from the archaebacterium Methanobacterium thermoautotrophicum, the first example from an anaerobic bacterium. The deduced amino acid sequence showed overall similarity to sequences of known Mn- and Fe-SODs from aerobic organisms. Judging from a detailed sequence comparison, the cloned SOD gene is classified as Mn-SOD. By comparison of Mn-SOD sequences among various species it was suggested that archaebacterial superoxide dismutase is a direct descendant of a primordial enzyme. Between a putative promoter and the start codon there is an inverted repeat sequence which is also found in the counterpart of Halobacterium halobium.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.