Upgrade of wood sugar d-xylose to a value-added nutraceutical by in vitro metabolic engineering.

The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.

Glycerol Phosphate Cytidylyltransferase Stereospecificity Is Key to Understanding the Distinct Stereochemical Compositions of Glycerophosphoinositol in Bacteria and Archaea.

Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in Bacteria and Archaea, and the basis for this domain-dependent specificity was investigated herein. The archaeon Archaeoglobus fulgidus and the bacterium Aquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and l-myo-inositol 1-phosphate. DIPPS of A. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of 31P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of A. fulgidus and Aq. aeolicus, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for sn-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for sn-glycerol 3-phosphate. In contrast, the partially purified A. fulgidus GCT uses sn-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e., A. fulgidus accumulates predominantly sn-glycero-1-phospho-3-l-myo-inositol, while Aq. aeolicus accumulates sn-glycero-3-phospho-3-l-myo-inositol. IMPORTANCE: Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and l-myo-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the Bacteria and Archaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require sn-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.

Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme.

Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.

Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.

Enzyme-driven speciation: crystallizing Archaea via lipid capture.

As the origin(s) of life on Earth remains an open question, detailed characteristics about the "last universal ancestor" (LUA) continue to be obscured. Here we provide arguments that strengthen the bacterial-like nature of the LUA. Our view attempts to recreate the evolution of archaeal lipids, the major components of the distinctive membrane that encapsulates these ancient prokaryotes. We show that (S)- 3-O-geranylgeranylglyceryl phosphate synthase (GGGPS), a TIM-barrel protein that performs the committed step in archaeal lipid synthesis, likely evolved from the duplication and fusion of a (betaalpha)4 half-barrel ancestor. By comparison to the well-characterized HisA and HisF TIM-barrel proteins, we propose a time line for the invention of this diagnostic archaeal biosynthetic pathway. After excluding the possibility of horizontal gene transfer, we conclude that the evolutionary history of GGGPS mirrors the emergence of Archaea from the LUA. We illustrate aspects of this "lipid capture" model that support its likelihood in recreating key evolutionary events and, as our hypothesis is built on a single initiating event, we suggest that the appearance of GGGPS represents an example of enzyme-driven speciation.

Characterization of AMA, a new AAA protein from Archaeoglobus and methanogenic archaea.

We have previously reported a new group of AAA proteins, which is only found in Archaeoglobus and methanogenic archaea (AMA). The proteins are phylogenetically basal to the metalloprotease clade and their N-terminal domain is homologous to the beta-clam part of the N-domain of CDC48-like proteins. Here we report the biochemical and biophysical characterization of Archaeoglobus fulgidus AMA, and of its isolated N-terminal (AMA-N) and ATPase (AMA-DeltaN) domains. AfAMA forms hexameric complexes, as does AMA-N, while AMA-DeltaN only forms dimers. The ability to hexamerize is dependent on the integrity of a GYPL motif in AMA-N, which resembles the pore motif of FtsH and HslU. While the physiological function of AMA is unknown, we show that it has ATP-dependent chaperone activity and can prevent the thermal aggregation of proteins in vitro. The ability to interact with non-native proteins resides in the N-domain and is energy-independent.

The putative lipase, AF1763, from Archaeoglobus fulgidusis is a carboxylesterase with a very high pH optimum.

The open reading frame AF1763, annotated as a putative lipase gene (lipA) of the hyperthermophilic archaeon, Archaeoglobus fulgidus DSM 4304, was cloned and over-expressed in E. coli. A sequence analysis of LipA and the investigation of a truncated enzyme implied a special function of the C-terminal part of LipA. The substrate spectrum of the enzyme suggested that LipA is a carboxylesterase rather than a canonical lipase. The enzyme showed optimal activity at 70 degrees C and between pH 10 and 11, which is among the most alkaline pH range detected for hydrolases.

Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus.

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. Two types of Hdr have been identified and characterized from distantly related methanogens. Here we show that the sulfate-reducing archaeon Archaeoglobus profundus cultivated on H2/sulfate forms enzymes related to both types of Hdr. From the membrane fraction of A. profundus, a two-subunit enzyme (HmeCD) composed of a b-type cytochrome and a hydrophilic iron-sulfur protein was isolated. The amino-terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A. fulgidus. HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp. From the soluble fraction of A. profundus a six-subunit enzyme complex (Mvh:Hdl) containing Ni, iron-sulfur clusters and FAD was isolated. Via amino-terminal sequencing, the encoding genes were identified in the genome of the closely related species A. fulgidus in which these genes are clustered. They encode a three-subunit [NiFe] hydrogenase with high sequence identity to the F420-nonreducing hydrogenase from Methanothermobacter spp. while the remaining three polypeptides are related to the three-subunit heterodisulfide reductase from Methanothermobacter spp. The oxidized enzyme exhibited an unusual EPR spectrum with gxyz = 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr. Upon reduction with H2 this signal was no longer detectable.

Denaturing action of urea and guanidine hydrochloride towards two thermophilic esterases.

The stability of two thermophilic esterases, AFEST from Archaeoglobus fulgidus and EST2 from Alicyclobacillus acidocaldarius, against the denaturing action of urea and guanidine hydrochloride has been investigated by means of steady-state fluorescence and circular dichroism measurements. Experimental results indicate that the two enzymes, even though very resistant to temperature and urea, show a resistance to guanidine hydrochloride weaker than expected on the basis of data collected so far for a large set of globular proteins. Structural information available for AFEST and EST2 and ideas that emerged from studies on the molecular origin of the greater thermal stability of thermophiles allow the suggestion of a reliable rationale. The present results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for the stability of the two esterases.

Purification and characterization of a membrane-bound enzyme complex from the sulfate-reducing archaeon Archaeoglobus fulgidus related to heterodisulfide reductase from methanogenic archaea.

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. The genome of the sulfate-reducing archaeon Archaeoglobus fulgidus encodes several proteins of unknown function with high sequence similarity to the catalytic subunit of Hdr. Here we report on the purification of a multisubunit membrane-bound enzyme complex from A. fulgidus that contains a subunit related to the catalytic subunit of Hdr. The purified enzyme is a heme/iron-sulfur protein, as deduced by UV/Vis spectroscopy, EPR spectroscopy, and the primary structure. It is composed of four different subunits encoded by a putative transcription unit (AF499, AF501-AF503). A fifth protein (AF500) encoded by this transcription unit could not be detected in the purified enzyme preparation. Subunit AF502 is closely related to the catalytic subunit HdrD of Hdr from Methanosarcina barkeri. AF501 encodes a membrane-integral cytochrome, and AF500 encodes a second integral membrane protein. AF499 encodes an extracytoplasmic iron-sulfur protein, and AF503 encodes an extracytoplasmic c-type cytochrome with three heme c-binding motifs. All of the subunits show high sequence similarity to proteins encoded by the dsr locus of Allochromatium vinosum and to subunits of the Hmc complex from Desulfovibrio vulgaris. The heme groups of the enzyme are rapidly reduced by reduced 2,3-dimethyl-1,4-naphthoquinone (DMNH2), which indicates that the enzyme functions as a menaquinol-acceptor oxidoreductase. The physiological electron acceptor has not yet been identified. Redox titrations monitored by EPR spectroscopy were carried out to characterize the iron-sulfur clusters of the enzyme. In addition to EPR signals due to [4Fe-4S]+ clusters, signals of an unusual paramagnetic species with g values of 2.031, 1.994, and 1.951 were obtained. The paramagnetic species could be reduced in a one-electron transfer reaction, but could not be further oxidized, and shows EPR properties similar to those of a paramagnetic species recently identified in Hdr. In Hdr this paramagnetic species is specifically induced by the substrates of the enzyme and is thought to be an intermediate of the catalytic cycle. Hence, Hdr and the A. fulgidus enzyme not only share sequence similarity, but may also have a similar active site and a similar catalytic function.

Biochemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii.

A novel dNTP pyrophosphatase, Mj0226 from Methanococcus jannaschii, which catalyzes the hydrolysis of nucleoside triphosphates to the monophosphate and PPi, has been characterized. Mj0226 protein catalyzes hydrolysis of two major substrates, dITP and XTP, suggesting that the 6-keto group of hypoxanthine and xanthine is critical for interaction with the protein. Under optimal reaction conditions the k(ca)(t) /K(m) value for these substrates was approximately 10 000 times that with dATP. Neither endonuclease nor 3'-exonuclease activities were detected in this protein. Interestingly, dITP was efficiently inserted opposite a dC residue in a DNA template and four dNTPs were also incorporated opposite a hypoxanthine residue in template DNA by DNA polymerase I. Two protein homologs of Mj0226 from Escherichia coli and Archaeoglobus fulgidus were also cloned and purified. These have catalytic activities similar to Mj0226 protein under optimal conditions. The implications of these results have significance in understanding how homologous proteins, including Mj0226, act biologically in many organisms. It seems likely that Mj0226 and its homologs have a major role in preventing mutations caused by incorporation of dITP and XTP formed spontaneously in the nucleotide pool into DNA. This report is the first identification and functional characterization of an enzyme hydrolyzing non-canonical nucleotides, dITP and XTP.

A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V.

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.

Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase.

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 degrees C, deltaHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 microM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered K(M) and k(cat) values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.