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Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils., (© 2013.)

The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275., (Copyright © 2011 Elsevier B.V. All rights reserved.)

d-Arabinose isomerase (d-AI), also known as l-fucose isomerase (l-FI), catalyzes the aldose-ketose isomerization of d-arabinose to d-ribulose, and l-fucose to l-fuculose. Bacillus pallidus (B. pallidus) d-AI can catalyze isomerization of d-altrose to d-psicose, as well as d-arabinose and l-fucose. Three X-ray structures of B. pallidus d-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor, l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60 A, respectively. B. pallidus d-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (beta/alpha) barrel fold domain. A catalytic metal ion (Mn(2+)) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose-ketose isomerization by B. pallidus d-AI, as well as Escherichia coli (E. coli) l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidus d-AI possibly interconverts between "open" and "closed" forms, and that the additional metal ion found in B. pallidus d-AI may help to stabilize the channel region., (Copyright 2010 Elsevier B.V. All rights reserved.)

The methylerythritol phosphate pathway to isoprenoids has been firmly established as an alternate to the mevalonate pathway in many bacteria, plants, algae, and the malaria parasite Plasmodium falciparum. The second enzyme in this pathway, deoxy-D-xylulose 5-phosphate reductoisomerase (DXR; E.C. 1.1.1.267), has been the focus of many investigations since it was found to be the target of the antibacterial and antimalarial compound, fosmidomycin. Several x-ray crystal structures of the Escherichia coli and Zymomonas mobilis DXR enzymes have provided important structural information about the residues potentially involved in substrate binding and catalysis. Site-directed mutagenesis studies can be used to complement the structural studies, providing kinetic data for specific changes of active site residues. Active site mutants were prepared of the recombinant Synechocystis sp. PCC6803 DXR, targeting residues D152, S153, E154, H155, M206, and E223. Alteration of the three acidic residues had major effects on catalysis, changes to S153 and M206 had variable effects on binding and catalysis, and a H155A mutation had only minimal effects on the kinetic parameters.

We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-xylose. These findings explain the extremely low reaction rate of xylose isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-A resolution, and that of a binary complex with the co-substrate NADPH to 2.7-A resolution. The subunit of DXR consists of three domains. Residues 1-150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed beta-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300-386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after beta1 and with the loops between alpha2 and beta2 and alpha5 and beta5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.

The dxr gene encoding the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The K(m)'s for the metal ions Mn(2+), Mg(2+), and Co(2+) were determined for native DXR for the first time, with the K(m) for Mg(2+) being approximately 200-fold higher than the K(m)'s for Mn(2+) and Co(2+).

The gene encoding a novel L-ribose isomerase (L-RI) from Acinetobacter sp. was cloned into Escherichia coli and nucleotide sequence was determined. The gene corresponded to an open reading frame of 747 bp that codes for a deduced protein of 249 amino acids, which showed no amino acid sequence similarity with any other sugar isomerases. After expression of the gene in E. coli using pUC118 the recombinant L-RI was purified to homogeneity using different chromatographic methods. The overall enzymatic properties of the purified recombinant L-RI were the same as those of the authentic L-RI. To our knowledge, this is the first time report concerning the L-RI gene.

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.

Two periwinkle cDNAs (crdxr and crmecs) encoding enzymes of the non-mevalonate terpenoid pathway were characterized using reverse transcription-PCR strategy based on the design of degenerated oligonucleotides. The deduced amino acid sequence of crdxr is homologue to 1-deoxy-D-xylulose 5-phosphate reductoisomerases. Crmecs represents the first plant cDNA encoding a protein similar to the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Escherichia coli. Expression of crdxr and crmecs genes was up-regulated in periwinkle cells producing monoterpenoid indole alkaloids. Involvement of the 2C-methyl-D-erythritol 4-phosphate pathway in alkaloid biosynthesis is discussed.

We found a significant activity of hydroxypyruvate isomerase in Escherichia coli clone cells harboring an E. coli gene (called orf b0508 or gip), which is located downstream of the glyoxylate carboligase gene. We newly designated the gene hyi. The enzyme was purified from cell extracts of the E. coli clone. The enzyme had a molecular mass of 58 kDa and was composed of two identical subunits. The optimum pH for the isomerization of hydroxypyruvate was 6.8-7.2. The enzyme required no cofactor. It exclusively catalyzed the isomerization between hydroxypyruvate and tartronate semialdehyde. The apparent K(m) value for hydroxypyruvate was 12.5 mM. The amino acid sequence of E. coli hydroxypyruvate isomerase is highly similar to those of glyoxylate-induced proteins, Gip, found widely from prokaryotes to eukaryotes.

The enzyme glucosamine-6-phosphate deaminase from beef kidney has been purified to homogeneity by allosteric-site affinity chromatography. Its amino acid composition and the N-terminal sequence (1-42), were obtained. The amino acid sequence of this segment is essentially identical to the corresponding regions of the human and hamster glucosamine-6-phosphate deaminases. The beef enzyme is a hexamer of 32.5 kDa subunits; this is nearly 2.5 kDa higher than the molecular mass of the homologous enzyme from Escherichia coli. Beef kidney deaminase exhibits a notable difference from the bacterial enzyme in its allosteric activation by N-acetylglucosamine 6-phosphate This metabolite, which is also is the allosteric activator of the bacterial glucosamine-6-phosphate deaminase, activates the enzyme by increasing its kcat without any change in the Km values for glucosamine 6-phosphate, over a wide range of activator concentration. This observation places beef kidney deaminase in the class of V-type allosteric systems.