Aldose-ketose Isomerase Research Articles

Kinetic characterization of methylthio-d-ribose-1-phosphate isomerase.

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-d-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-d-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose-ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct.

Covalent Adduct Formation in Methylthio-d-ribose-1-phosphate Isomerase: Reaction Intermediate or Artifact?

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) functions in the methionine salvage pathway by converting the cyclic aldose MTR1P to its open-chain ketose isomer methylthio-d-ribulose 1-phosphate (MTRu1P). What is particularly challenging for this enzyme is that the substrate's phosphate ester prevents facile equilibration to an aldehyde, which in other aldose-ketose isomerases is known to activate the α-hydrogen for proton or hydride transfer between adjacent carbons. We speculated that MtnA could use covalent catalysis via a phosphorylated residue to permit isomerization by one of the canonical mechanisms, followed by phosphoryl transfer back to form the product. In apparent support of this mechanism, [32P]MTR1P was found by SDS-PAGE and gel-filtration chromatography to radiolabel the enzyme. Susceptibility of this adduct to strongly acidic and basic pH and nucleophilic agents is consistent with an acyl phosphate. C160S and D240N, mutants of two conserved active-site residues, however, exhibited no difference in radiolabeling despite a reduction in activity of ∼107, leading to the conclusion that phosphorylation is unrelated to catalysis. Unexpectedly, prolonged incubations with C160S revealed up to 30% accumulation of radioactivity, which was identified by 31P and 13C NMR to be the result of a second adduct─a hemiketal formed between Ser160 and the carbonyl of MTRu1P. These results are interpreted as indirect support for a mechanism involving transfer of the proton from C-2 to C-1 by Cys160.

Biochemical characterization of recombinant L-fucose isomerase from Caldanaerobius polysaccharolyticus for L-fuculose production

L-fuculose is a rare sugar that is useful for the agriculture and medicine industries. L-fucose isomerase (E.C.5.3.1.25), which is an aldose-ketose isomerase, plays a significant role in producing rare sugars. A recommended L-fucose isomerase gene was cloned from Caldanaerobius polysaccharolyticus and purified with a single band of 65 kDa using nickel-affinity chromatography, with a specific activity of 108.23 U mg−1. The native molecular mass existed with 214 kDa was a trimer. The purified enzyme showed a maximum activity in 1 mM Mn2+ at 55 °C and pH 6.5 with a melting temperature (Tm) of 80.3 °C in the presence of one molecule per monomer. L-fucose isomerase from C. polysaccharolyticus (Capo-LfIase) exhibited the highest activity of L-fucose with Km, kcat and Kcat/km values of 94.2 mM, 23854 min−1 and 253.3 min−1 mM−1, respectively. Capo-LfIase showed more than 50% thermostability after 20 h of incubation at 45, 55, 65, 75 and 85 °C. The 9 putative active site residues of the L-fucose substrate were described using a homology model, and the results showed that Tyr440, Met185, Trp499 and Asn527 are the candidates of metal-binding residues, while Ser393, Glu337, Glu302, His528 and Asp361 would be involved in substrate binding. The conversion rate of L-fuculose from L-fucose was almost 28.2%, with 80 g L−1 L-fucose, and no byproduct was found. To the best of our knowledge, Capo-LfIase produces high yield of L-fuculose from L-fucose by enzymatic methods.

Isomerases and epimerases for biotransformation of pentoses.

Pentoses represent monosaccharides with five carbon atoms. They are organized into two main groups, aldopentoses and ketopentoses. There are eight aldopentoses and four ketopentoses and each ketopentose corresponds to two aldopentoses. Only D-xylose, D-ribose, and L-arabinose are natural sugars, but others belong to rare sugars that occur in very small quantities in nature. Recently, rare pentoses attract much attention because of their great potentials for commercial applications, especially as precursors of many important medical drugs. Pentoses Izumoring strategy provides a complete enzymatic approach to link all pentoses using four types of enzymes, including ketose 3-epimerases, aldose-ketose isomerases, polyol dehydrogenases, and aldose reductases. At least 10 types of epimerases and isomerases have been used for biotransformation of all aldopentoses and ketopentoses, and these enzymes are reviewed in detail in this article.

D-lyxose isomerase and its application for functional sugar production.

Functional sugars have attracted attention because of their wide application prospects in the food, cosmetics, and pharmaceutical industries in recent decades. Compared with complex chemical synthesis, enzymatic methods of creating functional sugars, characterized by high specificity, moderate reaction conditions, and sustainability, are favored. D-lyxose isomerase (D-LI, EC 5.3.1.15), an important aldose-ketose isomerase, catalyzes the reverse isomerization reaction between D-xylulose and D-lyxose, as well as D-fructose and D-mannose. D-LI has drawn researchers' attention due to its broad substrate specificity and high potential for enzymatic production of some functional sugars such as D-xylulose, D-mannose, and D-ribose. In this article, an overview of recent advances in the biochemical properties of various D-LIs is explored in detail. Structural analysis, active site identification, and catalytic mechanisms are also provided. Additionally, the applications of D-LIs for functional sugar production, including D-lyxose, D-mannose, and L-ribose, are reviewed in detail in this paper.

Biochemical and structural characterization of Marinomonas mediterranead-mannose isomerase Marme_2490 phylogenetically distant from known enzymes

d-Mannose isomerase (MI) reversibly isomerizes d-mannose to d-fructose, and is attractive for producing d-mannose from inexpensive d-fructose. It belongs to the N-acylglucosamine 2-epimerase (AGE) superfamily along with AGE, cellobiose 2-epimerase (CE), and aldose-ketose isomerase (AKI). In this study, Marinomonas mediterranea Marme_2490, showing low sequence identity with any known enzymes, was found to isomerize d-mannose as its primary substrate. Marme_2490 also isomerized d-lyxose and 4-OH d-mannose derivatives (d-talose and 4-O-monosaccharyl-d-mannose). Its activity for d-lyxose is known in other d-mannose isomerizing enzymes, such as MI and AKI, but we identified, for the first time, its activity for 4-OH d-mannose derivatives. Marme_2490 did not isomerize d-glucose, as known MIs do not, while AKI isomerizes both d-mannose and d-glucose. Thus, Marme_2490 was concluded to be an MI.The initial and equilibrium reaction products were analyzed by NMR to illuminate mechanistic information regarding the Marme_2490 reaction. The analysis of the initial reaction product revealed that β-d-mannose was formed. In the analysis of the equilibrated reaction products in D2O, signals of 2-H of d-mannose and 1-H of d-fructose were clearly detected. This indicates that these protons are not substituted with deuterium from D2O and Marme_2490 catalyzes the intramolecular proton transfer between 1-C and 2-C.The crystal structure of Marme_2490 in a ligand-free form was determined and found that Marme_2490 is formed by an (α/α)6-barrel, which is commonly observed in AGE superfamily enzymes. Despite diverse reaction specificities, the orientations of residues involved in catalysis and substrate binding by Marme_2490 were similar to those in both AKI (Salmonella enterica AKI) and epimerase (Rhodothermus marinus CE). The Marme_2490 structure suggested that the α7→α8 and α11→α12 loops of the catalytic domain participated in the formation of an open substrate-binding site to provide sufficient space to bind 4-OH d-mannose derivatives.

Characterization of a thermostable recombinant l-rhamnose isomerase from Caldicellulosiruptor obsidiansis OB47 and its application for the production of l-fructose and l-rhamnulose.

l-Hexoses are rare sugars that are important components and precursors in the synthesis of biological compounds and pharmaceutical drugs. l-Rhamnose isomerase (L-RI, EC 5.3.1.14) is an aldose-ketose isomerase that plays a significant role in the production of l-sugars. In this study, a thermostable, l-sugar-producing L-RI from the hyperthermophile Caldicellulosiruptor obsidiansis OB47 was characterized. The recombinant L-RI displayed maximal activity at pH 8.0 and 85 °C and was significantly activated by Co2+ . It exhibited a relatively high thermostability, with measured half-lives of 24.75, 11.55, 4.15 and 3.30 h in the presence of Co2+ at 70, 75, 80 and 85 °C, respectively. Specific activities of 277.6, 57.9, 13.7 and 9.6 U mg-1 were measured when l-rhamnose, l-mannose, d-allose and l-fructose were used as substrates, respectively. l-Rhamnulose was produced with conversion ratios of 44.0% and 38.6% from 25 and 50 g L-1 l-rhamnose, respectively. l-Fructose was also efficiently produced by the L-RI, with conversion ratios of 67.0% and 58.4% from 25 and 50 g L-1 l-mannose, respectively. The recombinant L-RI could effectively catalyze the formation of l-rhamnulose and l-fructose, suggesting that it was a promising candidate for industrial production of l-rhamnulose and l-fructose. © 2017 Society of Chemical Industry.

Characterization of a novel thermostable l-rhamnose isomerase from Thermobacillus composti KWC4 and its application for production of d-allose

Abstract d -Allose was considered as a kind of rare sugars with testified potential medicinal and agricultural benefits. l -Rhamnose isomerase (L-RI, EC 5.3.1.14), an aldose-ketose isomerase, played a significant part in producing rare sugar. In this article, a thermostable d -allose-producing L-RI was characterized from a thermotolerant bacterium, Thermobacillus composti KWC4. The recombinant L-RI was activated obviously in the presence of Mn2+ with an optimal pH 7.5 and temperature 65 °C. The Michaelis-Menten constant (Km), turnover number (kcat) and catalytic efficiency (kcat/Km) for l -rhamnose were 33.8 mM, 1189.8 min−1 and 35.2 min−1 mM−1, respectively. At a higher temperature, Mn2+ played a pivotal role in strengthening the thermostability of T. composti L-RI. The differential scanning calorimetry (DSC) results showed the denaturing temperature (Tm) of T. composti L-RI was increased by 3 °C in presence of Mn2+. Although the T. composti L-RI displayed the optimum substrate as l -rhamnose, it could also effectively catalyze the isomerization between d -allulose and d -allose. When the reaction reached equilibrium, the sole product d -allose was produced from D-alluose by T. composti L-RI.

Characteristics and Kinetic Properties of L-Rhamnose Isomerase from <i>Bacillus Subtilis</i> by Isothermal Titration Calorimetry for the Production of D-Allose

L-Rhamnose isomerase, an aldose-ketose isomerase, is an important enzyme for rare sugar production. In this paper, L-rhamnose isomerase (L-RhI) from Bacillus subtilis 168 (B. subtilis L-RhI) was cloned and expressed in Bacillus subtilis WB600 for the production of D-allose. The obtained crude L-RhI was then purified through a HisTrap HP affinity chromatography column and an anion-exchange chromatography column. The characteristics of purified L-RhI were analyzed subsequently. The enzyme was activated in presence of Mn2+, and it had thermal advantages as its optimum temperature was 70°C, and 85% of its activity was retained after 4 hours' incubation at 60°C. In addition, isothermal titration calorimetry (ITC) was for the first time employed to determine the isomerization of rare sugars, and the kinetic data showed that B. subtilis L-RhI had relatively low Km values on the substrates of L-rhamnose, L-mannose, D-ribose and D-allose. Using D-psicose as the substrate, no by-products but only D-allose was generated catalyzed by B. subtilis L-RhI, and the yield of 37.51% was obtained when the reaction reached its equilibrium at 60°C.

Overexpression, crystallization and preliminary X-ray crystallographic analysis ofD-ribose-5-phosphate isomerase fromClostridium thermocellum

Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate isomerases. Recently, Clostridium thermocellum d-ribose-5-phosphate isomerase (CTRPI), an aldose-ketose isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 69.5, c = 154.4 angstrom, and diffracted to 1.9 angstrom resolution. According to Matthews coefficient calculations, the crystallographic structure consists of a dimer in the asymmetric unit, with a V(M) of 3.2 angstrom(3) Da(-1) and a solvent content of 61.7%.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Proton diffusion in the active site of triosephosphate isomerase

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallographic studies of the mechanism of xylose isomerase

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.

8] Enzymology of enol intermediates

Publisher Summary This chapter discusses reactions most commonly classified as stepwise processes with enol intermediates. The term “enol” is not meant to exclude the enolate as the functioning species except when the question is specifically addressed. There are many examples of multifunctional enzymes that are thought to use enols as intermediates: ribulose-1,5-P 2 carboxylase, glyoxalase, oxidativeβ-ketodecarboxylases, ketosteroid isomerase, and thymidylate synthetase. Examples of enzymes that use imine intermediates are known for all the reactions shown above except those that specifically call for an oxygen atom, such as the usual aldose-ketose isomerases, dehydration, and enol-kinase. Enols that are produced catalytically as normal products of enzymes may be used as a source to test as an intermediate of another enzyme. A more general approach, also biosynthetic, is to use the bound intermediate of an enzymic reaction as a source of the free form to be tested with another enzyme. This approach is promising when the source enzyme is available in adequate amount and requires that the intermediate be enriched on the enzyme above its expected unfavorable concentration in solution equilibrium. The enols that have been mentioned already, hydroxy maleate, p-OH-phenylenolpyruvate, and enolpyruvate, have sufficient stability to survive the operations of denaturation and neutralization, which at the fastest would require 5 msec.

An intramolecular C-2 → C-1 hydrogen transfer during the acid-catalyzed conversion of d-xylose to d- threo-pentulose ( d-xylulose)

Aldose-ketose isomerases are known to catalyze a partial and sometimes complete intramolecular hydrogen transfer between C-1 of the ketose and C-2 of the aldose. It was recently shown (Feather, M. S., and Harris, D. W. (1975) J. Amer. Chem. Soc. 97, 178–181) that the same type of transfer occurs during the acid-catalyzed interconversion of d-fructose, d-glucose, and d-mannose. A similar transfer is demonstrated herein for the conversion of d-xylose to d-xylulose in acid solution. d-[2- 3H]xylose was isomerized in aqueous sulfuric acid and the resulting d-[ 3H]xylulose was isolated in 6% yield. The ketose had 18.3% the activity of the starting aldose. Chemical degradation showed that all the carbon-bound tritium of the d-[ 3H]xylulose was located at C-1, thus indicating a C-2 → C-1 intramolecular hydrogen transfer. During the reaction, less than 1.2% of the total radiochemical activity was found in the solvent, and, the unreacted d-[2- 3H]xylose was recovered, having an activity nearly the same as the starting material. The differences in activity, therefore, of the d-[2- 3H]xylose and the d-[1- 3H]xylulose are due to an isotope effect ( K H K T ) which is indicated to be 5.4. The data are discussed in terms of currently accepted models for isomerase mechanisms.

Stereochemical evidence for a cis-enediol intermediate in Mn-dependent aldose isomerases

Three Mn 2+-dependent bacterial aldose-ketose isomerases were examined to determine the stereochemistry of the C-1 proton of the ketose that is abstracted in the formation of the aldehyde. In the cases of d-xylose isomerase ( d-xylose ketol-isomerase, EC 5.3.1.5) and l-arabinose isomerase ( l-arabinose ketol-isomerase, EC 5.3.1.4) it is the pro-R position of the d-xylulose and l-ribulose that is activated, whereas in the l-fucose isomerase reaction it is the pro-S position of l-fuculose that is labeled in tritiated water. Little proton exchange occurs in these reactions, especially the xylose isomerase, where retention of the substrate proton is complete. These stereochemical results conform to those of four other sugar isomerases: triose- P, d- ribose-5-P , d- mannose-6-P , and d- glucose-6-P isomerase. Protonation at C-1 or C-2 from the same side of the plane of a cis-enediol gives the correct stereochemical result for all seven isomerase.