Ribose-5-phosphate isomerases: characteristics, structural features, and applications.

Ribose-5-phosphate isomerase (Rpi, EC 5.3.1.6) is widespread in microorganisms, animals, and plants. It has a pivotal role in the pentose phosphate pathway and responsible for catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate. In recent years, Rpi has received considerable attention as a multipurpose biocatalyst for production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose. Besides, it has been thought of as a potential drug target in the treatment of trypanosomatid-caused diseases such as Chagas' disease, leishmaniasis, and human African trypanosomiasis. Despite increased research activities, up to now, no systematic review of Rpi has been published. To fill this gap, this paper provides detailed information about the enzymatic properties of various Rpis. Furthermore, structural features, catalytic mechanism, and molecular modifications of Rpis are summarized based on extensive crystal structure research. Additionally, the applications of Rpi in rare sugar production and the role of Rpi in trypanocidal drug design are reviewed. Key points • Fundamental properties of various ribose-5-phosphate isomerases (Rpis). • Differences in crystal structure and catalytic mechanism between RpiA and RpiB. • Application of Rpi as a rare sugar producer and a potential drug target.

Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O2 and O4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions.

Evolutionary History of the Enzymes Involved in the Calvin-Benson Cycle in Euglenids.

Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin-Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin-Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin-Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.

Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating <90% identity with known XIs in the database accounted for 89% of the total xylA phylotypes. The differences among xylA members and compositions within each soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications.

Computational analysis of the evolution of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase, an important enzyme in plant terpene biosynthesis.

Isoprenoids are a highly diverse and important group of natural compounds. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) catalyzes a key regulatory step in the non-mevalonate isoprenoid biosynthetic pathway in eubacteria and in plant plastids. For example, in Artemisia annua DXR participates in regulation of the biosynthesis of artemisinin, an important antimalarial drug. We performed phylogenetic analysis using DXR protein sequences from a model prokaryote, Escherichia coli, a picoplanktonic alga, Ostreococcus lucimarinus, and higher plants. The functional domain of DXR was conserved, allowing molecular evolutionary comparisons of both prokaryotic and eukaryotic sequences of DXR. Despite this conservation, for some plant species such as Campthoteca acuminata and Arabidopsis thaliana, phylogenetic relationships of their lineages were consistently violated. Our analysis revealed that plant DXR has an N-terminal transit domain that is likely bipartite, consisting of a chloroplast transit peptide (cTP) and a lumen transit peptide (lTP). Several features observed in the lTP suggest that, while DXR is targeted to the chloroplast, it is localized to the thylakoid lumen. These features include a twin arginine motif, a hydrophobic region, and a proline-rich region. The transit peptide also showed putative motifs for a 14-3-3 binding site with a chaperone phosphorylation site at Thr.

Biosynthesis of isoprenoids: studies on the mechanism of 2C-methyl-D-erythritol-4-phosphate synthase.

2C-Methyl-D-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1-deoxy-D-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-D-erythritol 4-phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol-aldol sequence. We report the preparation of (13)C-labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol-aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-D-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1-(13)C(1)]-2C-methyl-D-erythritol 4-phosphate and [3-(13)C(1)]2C-methyl-D-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-D-xylulose 5-phosphate to give 1-deoxy-L-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol-aldol mechanism cannot be ruled out conclusively, the data show that a retroldol-aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.

Glucose-to-fructose conversion at high temperatures with xylose (glucose) isomerases from Streptomyces murinus and two hyperthermophilic Thermotoga species.

The conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg(2+), the temperature optima for the three soluble enzymes were 85 degrees C (SMGI), 95 degrees to 100 degrees C (TNGI), and >100 degrees C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first-order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg(2+). The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60 degrees C and 70 degrees C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg(2+) occurred at 70 degrees C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80 degrees C and in the presence of Mg(2+), productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose-fructose equilibrium, and enzyme inactivation, was used to examine the potential for high-fructose corn syrup (HFCS) production at 80 degrees C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co(2+) and Mg(2+). In the presence of both cations, these enzymes showed the potential to catalyze glucose-to-fructose conversion at 80 degrees C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55 degrees to 65 degrees C, but with the additional advantage of higher fructose concentrations. At 90 degrees C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies.

Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase.

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 degrees C, which is significantly higher than the optimal growth temperature (37 degrees C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 degrees C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme approximately Mn2+-enzyme, with melting temperatures of 50.3 degrees C, 53.3 degrees C, 73.4 degrees C, and 73.6 degrees C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ x mol(-1) (apoenzyme) to 604 kJ x mol(-1) (Mg2+-enzyme) to 1166 kJ x mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable alpha-amylase from B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone.

Glucose isomerase: insights into protein engineering for increased thermostability.

Thermostable glucose isomerases are desirable for production of 55% fructose syrups at >90 degrees C. Current commercial enzymes operate only at 60 degrees C to produce 45% fructose syrups. Protein engineering to construct more stable enzymes has so far been relatively unsuccessful, so this review focuses on elucidation of the thermal inactivation pathway as a future guide. The primary and tertiary structures of 11 Class 1 and 20 Class 2 enzymes are compared. Within each class the structures are almost identical and sequence differences are few. Structural differences between Class 1 and Class 2 are less than previously surmised. The thermostabilities of Class 1 enzymes are essentially identical, in contrast to previous reports, but in Class 2 they vary widely. In each class, thermal inactivation proceeds via the tetrameric apoenzyme, so metal ion affinity dominates thermostability. In Class 1 enzymes, subunit dissociation is not involved, but there is an irreversible conformational change in the apoenzyme leading to a more thermostable inactive tetramer. This may be linked to reversible conformational changes in the apoenzyme at alkaline pH arising from electrostatic repulsions in the active site, which break a buried Arg-30-Asp-299 salt bridge and bring Arg-30 to the surface. There is a different salt bridge in Class 2 enzymes, which might explain their varying thermostability. Previous protein engineering results are reviewed in light of these insights.