Identification and characterization of a L-tyrosine decarboxylase in Methanocaldococcus jannaschii

Methanofuran is the first coenzyme in the methanogenic pathway used by the archaeon Methanocaldococcus jannaschii, as well as other methanogens, to reduce CO2 to methane. The details of the pathway for the biosynthesis of methanofuran and the responsible genes have yet to be established. A clear structural element in all known methanofurans is tyramine, likely produced by the decarboxylation of L-tyrosine. We show here that the mfnA gene at M. jannaschii locus MJ0050 encodes a thermostable pyridoxal phosphate-dependent L-tyrosine decarboxylase that specifically produces tyramine. Homologs of this gene are widely distributed among euryarchaea but are not specifically related to known bacterial or plant tyrosine decarboxylases.     

Transaldolase of Methanocaldococcus jannaschii

The Methanocaldococcus jannaschii genome contains putative genes for all four nonoxidative pentose phosphate pathway enzymes. Open reading frame (ORF) MJ0960 is a member of the mipB/talC family of 'transaldolase-like' genes, so named because of their similarity to the well-characterized transaldolase B gene family. However, recently, it has been reported that both the mipB and the talC genes from Escherichia coli encode novel enzymes with fructose-6-phosphate aldolase activity, not transaldolase activity (Schürmann and Sprenger 2001). The same study reports that other members of the mipB/talC family appear to encode transaldolases. To confirm the function of MJ0960 and to clarify the presence of a nonoxidative pentose phosphate pathway in M. jannaschii, we have cloned ORF MJ0960 from M. jannaschii genomic DNA and purified the recombinant protein. MJ0960 encodes a transaldolase and displays no fructose-6-phosphate aldolase activity. It etained full activity for 4 h at 80 degrees C, and for 3 weeks at 25 degrees C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (Vmax) of 1.0 +/- 0.2 micromol min(-1) mg(-1) at 25 degrees C, whereas Vmax = 12.0 +/- 0.5 micromol min(-1) mg(-1) at 50 degrees C. Apparent Michaelis constants at 50 degrees C were Km = 0.65 +/- 0.09 mM for fructose-6-phosphate and Km = 27.8 +/- 4.3 microM for erythrose-4-phosphate. When ribose-5-phosphate replaced erythrose-4-phosphate as an aldose acceptor, Vmax decreased twofold, whereas the Km was 150-fold higher. The molecular mass of the active enzyme is 271 +/- 27 kDa as estimated by gel filtration, whereas the predicted monomer size is 23.96 kDa, suggesting that the native form of the protein is probably a decamer. A readily available source of thermophilic pentose phosphate pathway enzymes including transaldolase may have direct application in enzymatic biohydrogen production.     

Biochemical origins of lactaldehyde and hydroxyacetone in Methanocaldococcus jannaschii

The biochemical routes for the metabolism of methylglyoxal and the formation of lactaldehyde and hydroxyacetone in Methanocaldococcus jannaschii have been established. The addition of methylglyoxal and NADH, NADPH, F 420H 2, or DTT to a M. jannaschii cell extract stimulated the production of both lactaldehyde and hydroxyacetone. Using appropriately labeled NADH, NADPH, and F 420H 2, hydride transfer was only observed from F 420H 2 to lactaldehyde. It was shown that cell extracts of this Archaea readily catalyzed the F 420H 2-dependent reduction of methylglyoxal to lactaldehyde, a precursor of the lactate found in coenzyme F 420. This conversion was established by measuring the incorporation of deuterium from (5 RS)[5- (2)H 1]F 420H 2 into the C-2 position of the formed lactaldehyde. In vivo generated (5 R)[5- (2)H 1]F 420H 2 was also found to incorporate deuterium into lactaldehyde. The experimental data indicated that the pro- R hydrogen of F 420H 2 was transferred during the reduction. The stereochemistry of this transfer was opposite from that observed for all other known enzyme-catalyzed hydride-transfer reactions involving F 420. [1,3,3,3- (2)H 4]-Methylglyoxal was incorporated into lactaldehyde and hydroxyacetone as an intact unit during this reduction with the occurrence of some deuterium exchange. The exchange observed during this incorporation into lactaldehyde was substantially more than the exchange observed during the incorporation into the hydroxyacetone. The hydroxyacetone was derived directly from methylglyoxal, with the hydrogen for the reduction being derived from water. Hydroxyacetone was also readily formed by the condensation of pyruvate with formaldehyde. The product of the MJ0663 gene was shown to catalyze this condensation reaction.     

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii

Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12–16 nucleotides upstream of the corresponding translation start site. Analyses with 3′-RACE (rapid amplification of cDNA ends) and 5′-RACE indicate that the processing is endonucleolytic. Analyses of the sequences surrounding the processing sites for functional sites, sequence motifs, or potential RNA secondary structure elements did not reveal any recurring features except for an AUG translation start codon and (in most cases) a ribosome binding site. These properties differ from those of all previously described mRNA processing systems. Our data suggest that the processing alters the representation of various genes in the RNA pool and therefore, may play a significant role in defining the balance of proteins in the cell.     

The Methanocaldococcus jannaschii protein Mj0968 is not a P-type ATPase

The Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) protein Mj0968 has been reported to represent a soluble P-type ATPase [Ogawa et al., FEBS Lett. 471 (2000) 99-102]. In this study, we report that the heterologously expressed Mj0968-His(10) protein exhibits high rates of phosphatase activity, whereas only very low ATPase activity was measured. Replacement of the aspartate residue in the DSAGT motif (D7A), which becomes phosphorylated during the reaction cycle of P-type ATPases, does not affect the V(max), but only the K(M) of the reaction. Labeling studies with [gamma-(32)P]ATP and [alpha-(32)P]ATP revealed that the previously reported labeling experiments [Ogawa et al., 2000] do not necessarily show phosphorylation of Mj0968, but rather point to ATP binding. Binding studies with trinitrophenyl adenosine nucleotides showed low apparent K(d) values for those molecules. These results provide evidence that the native function of Mj0968 seems to be that of a phosphatase, rather than that of an ATP-hydrolyzing enzyme.     

Methanocaldococcus jannaschii uses a modified mevalonate pathway for biosynthesis of isopentenyl diphosphate

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the "missing" phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.     

Identification of lactaldehyde dehydrogenase in Methanocaldococcus jannaschii and its involvement in production of lactate for F420 biosynthesis

One of the early steps in the biosynthesis of coenzyme F(420) in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NAD(+)-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.     

Biochemical Characterization of a Dihydroneopterin Aldolase Used for Methanopterin Biosynthesis in Methanogens

The gene encoding 7,8-dihydroneopterin aldolase (DHNA) was recently identified in archaea through comparative genomics as being involved in methanopterin biosynthesis (V. Crécy-Lagard, G. Phillips, L. L. Grochowski, B. El Yacoubi, F. Jenney, M. W. Adams, A. G. Murzin, and R. H. White, ACS Chem. Biol. 7:1807–1816, 2012, doi:10.1021/cb300342u). Archaeal DHNA shows a unique secondary and quaternary structure compared with bacterial and plant DHNAs. Here, we report a detailed biochemical examination of DHNA from the methanogen Methanocaldococcus jannaschii. Kinetic studies show that M. jannaschii DHNA possesses a catalytic capability with a k_cat/K_m above 10⁵ M⁻¹ s⁻¹ at 70°C, and at room temperature it exhibits a turnover number (0.07 s⁻¹) comparable to bacterial DHNAs. We also found that this enzyme follows an acid-base catalytic mechanism similar to the bacterial DHNAs, except when using alternative catalytic residues. We propose that in the absence of lysine, which is considered to be the general base in bacterial DHNAs, an invariant water molecule likely functions as the catalytic base, and the strictly conserved His35 and Gln61 residues serve as the hydrogen bond partners to adjust the basicity of the water molecule. Indeed, substitution of either His35 or Gln61 causes a 20-fold decrease in k_cat. An invariant Tyr78 is also shown to be important for catalysis, likely functioning as a general acid. Glu25 plays an important role in substrate binding, since replacing Glu25 by Gln caused a ≥25-fold increase in K_m. These results provide important insights into the catalytic mechanism of archaeal DHNAs.     

Radical S-Adenosylmethionine (SAM) Enzymes in Cofactor Biosynthesis: A Treasure Trove of Complex Organic Radical Rearrangement Reactions

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F₄₂₀, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry.     

Ribose-5-phosphate biosynthesis in Methanocaldococcus jannaschii occurs in the absence of a pentose-phosphate pathway

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-2H2]- and [U-13C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-ara-bino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.     

詹氏甲烷球菌DNA连接酶在大肠杆菌中的表达纯化及连接活性鉴定

对一种耐热性古茵--詹氏甲烷球茵(Methanocaldococcus jannaschii)的DNA连接酶进行了克隆、表达、纯化,并对其生物化学特性和酶学活性进行了初步研究.詹氏甲烷球菌DNA连接酶重组蛋白在ATP及Mg<'2+>二价阳离子存在的条件下具有连接酶活性,能够封闭DNA链上的切割.通过不同温度下的测试,50～80℃为较适合连接温度,其耐热性强,甚至在90℃下加热5 min后仍有连接酶活性;其发挥活性的pH值范围比较宽泛.最适pH值为6.0～9.0.这是国际上对詹氏甲烷球菌DNA连接酶的首次报导.     

The Biosynthesis of Thiol- and Thioether-containing Cofactors and Secondary Metabolites Catalyzed by Radical S-Adenosylmethionine Enzymes

Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-l-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5′-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster''s endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products.     

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a k_cat/K_M of 3.2 × 10⁷ and 3.0 × 10⁷ s^{? 1}M^{? 1}, respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site‐directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10‐fold decrease in k_cat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828–840. © 2016 Wiley Periodicals, Inc.     

Enzymology and evolution of the pyruvate pathway to 2-oxobutyrate in Methanocaldococcus jannaschii

The archaeon Methanocaldococcus jannaschii uses three different 2-oxoacid elongation pathways, which extend the chain length of precursors in leucine, isoleucine, and coenzyme B biosyntheses. In each of these pathways an aconitase-type hydrolyase catalyzes an hydroxyacid isomerization reaction. The genome sequence of M. jannaschii encodes two homologs of each large and small subunit that forms the hydrolyase, but the genes are not cotranscribed. The genes are more similar to each other than to previously characterized isopropylmalate isomerase or homoaconitase enzyme genes. To identify the functions of these homologs, the four combinations of subunits were heterologously expressed in Escherichia coli, purified, and reconstituted to generate the iron-sulfur center of the holoenzyme. Only the combination of MJ0499 and MJ1277 proteins catalyzed isopropylmalate and citramalate isomerization reactions. This pair also catalyzed hydration half-reactions using citraconate and maleate. Another broad-specificity enzyme, isopropylmalate dehydrogenase (MJ0720), catalyzed the oxidative decarboxylation of beta-isopropylmalate, beta-methylmalate, and d-malate. Combined with these results, phylogenetic analysis suggests that the pyruvate pathway to 2-oxobutyrate (an alternative to threonine dehydratase in isoleucine biosynthesis) evolved several times in bacteria and archaea. The enzymes in the isopropylmalate pathway of leucine biosynthesis facilitated the evolution of 2-oxobutyrate biosynthesis through the introduction of a citramalate synthase, either by gene recruitment or gene duplication and functional divergence.     

Role of S-Adenosylmethionine in Methionine Biosynthesis in Yeast

Extracts of Saccharomyces cerevisiae were used to develop a cell-free system capable of converting the beta-carbon of serine into the methyl group of methionine. No requirement for either S-adenosylmethionine or S-adenosylhomocysteine could be demonstrated for net methionine biosynthesis. Growth of the cells in B(12) did not affect the reaction. The mechanism for the methylation of homocysteine in yeast appears to be similar to the non-B(12) system in Escherichia coli.     

Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.     

Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P,a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.