Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Kinetics and mathematical model of hydrolysis and transglycosylation catalysed by cellobiase

The kinetics of hydrolysis and transglycosylation reactions catalysed by cellobiase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus foetidus in the cellobiose-d-glucose reaction system have been studied. The formation of transglycosylation products was observed at cellobiose concentrations $\text{>10}{}^{\mathrm{?2}}\text{m}$, whereas at lower substrate concentrations the only reaction product was d-glucose. In the cellobiase-catalysed transglycosylation a (1→6)-β-linkage was formed after the transfer of a d-glucose residue to acceptor molecule. The basic transglycosylation products were isocellotriose and gentiobiose. A small amount of oligosaccharides with a higher degree of polymerization was also formed. The maximum content of transglycosylation products amounted to 25–30% of the total saccharide content in the system at the initial cellobiose concentration (0.1–0.3 m). The processes in the reaction system were inhibited by the substrate and product (d-glucose). A general scheme for cellobiose hydrolysis has been proposed and validated, allowing for the inhibition and transglycosylation effects. Based on this scheme, a mathematical model for cellobiose hydrolysis has been suggested to describe the kinetics of substrate consumption and product (d-glucose) accumulation, as well as the kinetics of formation and consumption of transglycosylation products throughout the course of enzymatic reaction with various initial amounts of cellobiose, starting from low concentrations up to 0.2–0.3 m (7–11% bv weight).     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into α-d-glucose 1-phosphate (G1P) and d-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites −1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy ⁴C₁ conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from ¹S₃ as the pre-transition state conformer (Michaelis complex) via E₃ as the transition state candidate to ⁴C₁ as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The ¹S₃ pre-transition state conformer is highly stable compared with other conformers, and a conformational change from ⁴C₁ to ^1,4B was observed.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Action of porcine-pancreatic amylase on oxidized-reduced amylose of low degree of modification

Amylose was oxidized with 0.1–0.2 mol of periodate per glucose residue (G), and then reduced with sodium borohydride or borotritide to give an oxidized-reduced amylose of low degree of modification. Mild acid hydrolysis gave erythritol, 2-O-α-d-glucosyl-l-erythritol, higher homologs, and other products. Extensive action of porcine-pancreatic amylase on the polymer gave, besides d-glucose and maltose, oligosaccharides containing one or more oxidized-reduced (modified, M), acyclic residues. The enzymic products containing only one oxidized-reduced residue were identified as a modified tetrasaccharide (MG₃) and a modified pentasaccharide (MG₄). Structures of MG₃ and MG₄ were identified by a combination of enzymic and chemical approaches. With glucoamylase, MG₄ was converted into MG plus d-glucose, whereas MG₃ was totally resistant. On mild acidic hydrolysis, MG₃ was converted into 2-O-α-d-glucosyl-d-erythritol plus maltose. These results indicate that MG₃ is G-M-G-G and that MG₄ is G-G-M-G-G. In principle, MG₄ could occupy the five d-glucose residue, substrate-binding site of porcine-pancreatic amylase in such a way that M, the acyclic structure replacing a d-glucose residue, is placed just to the “left” of the catalytic site. The modified structure, being very vulnerable to acidic hydrolysis, might then be expected to be very readily attacked by the amylase, but in fact, it is not.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

Purification and characterization of two β-glucosidases from a thermo-tolerant yeast Pichia etchellsii

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible β-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0–9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl β-d-glucopyranoside and alkyl β-d-glucosides whereas BGLII was most active on cellobiose. k_cat and K_M values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl-glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C₁₀) using dimethyl sulfoxide stabilized single-phase reaction microenvironment. About 12% molar yield of octyl-glucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned β-glucosidases from this yeast.     

Facile synthesis of 1,2-trans-O-acetyl glycosyl chloride derivatives of cellobiose,lactose, and D-glucose

Solutions of O-acetyl-α-glycosyl bromide derivatives of d-glucose, cellobiose, and lactose in hexamethylphosphoramide were converted into corresponding β-chlorides at room temperature by the action of lithium chloride. At 3:1 mM ratios of chloride ion to glycose, 5–10% w/v solutions of glycosyl bromide formed α- and β-chlorides in ratios of (or greater than) 1:19 within 2–13 min and produced crystalline β-chlorides in 70–80% yields. Anomeric compositions were determined by n.m.r. spectroscopy in hexamethylphosphoramide. Older methods of preparing 1,2-trans-O-acetylgIycosyl chlorides, with aluminum chloride or titanium tetrachloride, gave the α- and β-cellobiosyl and -Iactosyl chlorides in ratios that varied from 2:3 to 1:4 and reached 85–95% levels of β-chloride only with β-d-glucose pentaacetate. When hydrolyzed under conditions that controlled solution acidity, the β-cellobiosyl and -Iactosyl chlorides each gave 2-hydroxy derivatives in yields that could be varied from 16 to 60%. Hepta-O-acetyl-2-O-methyl-α-cellobiose was prepared to demonstrate how these hydrolysis mixtures can be used to synthesize a 2-O-substituted derivative.     

Synthesis and hydrolysis if N,N′-diglycopyranosylethyl-enediamines

Condensation products from ethylenediamine and simple reducing sugars were prepared and their chemical behavior was studied in order to understand how ethylenediamine may react with the reducing ends of wood polysaccharides in ethylenediamine-soda pulping. The N,N′-diglycosyldiamines containing d-glucose, d-mannose, and cellobiose were hydrolyzed rapidly but not completely in water at moderate-temperatures (35–65°). The reactions were at equilibrium; the greater the diamine concentration in solution, the less hydrolysis of the condensation products occurred.     

Cytochalasin B binding to Ehrlich ascites tumor cells and its relationship to glucose carrier

Cultured Ehrlich ascites tumor cells equilibrate d-glucose via a carrier mechanism with a K_m and V of 14 mM and 3 μmol/s per ml cells, respectively. Cytochalasin B competitively inhibits this carrier-mediated glycose transport with an inhibition constant (K_i) of approx. 5·10^?7 M. Cytochalasin E does not inhibit this carrier function. With cytochalasin B concentrations up to 1·10^?5 M, the range where the inhibition develops to practical completion, three discrete cytochalasin B binding sites, namely L, M and H, are distinguished. The cytochalasin B binding at L site shows a dissociation constant (K_d) of approx. 1·10^-6 M, represents about 30% of the total cytochalasin B binding of the cell (8·10⁶ molecules/cell), is sensitively displaced by cytochalasin E but not by d-glucose, and is located in cytosol. The cytochalasin B binding to M site shows a K_d of 4–6·10^?7 M, represents approx. 60% of the total saturable binding (14·10⁶ molecules/cell), is specifically displaced by d-glucose with a displacement constant of 15 mM, but not by l-glucose, and is insensitive to cytochalasin E. The sites are membrane-bound and extractable with Triton X-100 but not by EDTA in alkaline pH. The cytochalasin B binding at H site shows a K_d of 2–6 · 10^?8 M, represents less than 10% of the total sites (2 · 10⁶ molecules/cell), is not affected by either glucose or cytochalasin E and is of non-cytosol origin. It is concluded that the cytochalasin B binding at M site is responsible for the glucose carrier inhibition by cytochalasin B and the Ehrlich ascites cell is unique among other animal cells in its high content of this site. Approx. 16-fold purification of this site has been achieved.     

Affinity purification and glucose specificity of aldose reductase from bovine lens

Aldose reductase, a possible key enzyme of sugar-cataract formation in diabetes, has been purified from bovine lens by a five-step procedure including affinity chromatography with Mātrex gel red A. The enzyme was purified 12,600-fold and was apparently homogeneous by polyacrylamide gel electrophoresis. The glucose specificity of the purified enzyme was studied with d-glucose anomers and d-glucitol as substrates. The ratios of the reduction rate of α-d-glucose to that of β-d-glucose at 10, 13, and 20 mm were 1.90, 1.76, and 1.72, respectively. These values were in good agreement with the ratios (1.92, 1.81, and 1.66) calculated on the basis of the rate constants reported for d-glucose mutarotation equilibrium (J. M. Los, L. B. Simpson, and K. Wiesner, 1956, J. Amer. Chem. Soc.78, 1564–1568) and the assumption that aldose reductase acts on the aldehyde form of d-glucose. In addition, the composition of d-glucose produced from d-glucitol in the reverse reaction was 63% α anomer and 37% β anomer, which also agreed well with the values, 65 and 35%, respectively, calculated from the rate constants in reactions from the aldehyde form to both the α anomer and the β anomer. It was suggested from these kinetic analyses that aldose reductase acts on the aldehyde form of d-glucose (K_m = 0.66 μm) but not on either the α or the β anomer of d-glucose.     

A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes; its application to d-glucose transport studies in brush border vesicles from rabbit jejunum and ileum

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10–20 μl. Using this technique the kinetic parameters K_m and V of d-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the K_m for d-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Cellobiose Transport by Mixed Ruminal Bacteria from a Cow

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the K_m and 10 nmol/min/mg of protein for the V_max. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher V_max values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Endo-(1→4)-β-d-glucanases from sclerotium rolfsii. Purification,substrate specificity,and mode of action

Three purified endo-(1→4)-β-d-glucanases (EC 3.2.1.4), A, B, and C, from Sclerotium rolfsii culture filtrates showed homogeneity in disc-gel electrophoresis and in analytical isoelectric-focusing in polyacrylamide gel. The three endo-d-glucanases are glycoproteins, endo B and endo C being composed of a single polypeptide chain, and endo A of two dissimilar polypeptide chains that are covalently bound by a disulfide bridge. Endo B and endo C do not contain half-cystine residues. With carboxymethylcellulose as substrate, the liquifying activity of the three enzymes was inhibited by cellobiose but not by d-glucose. The specificity of the enzymes is restricted to β-(1→4) linkages, but they showed some differences in the mode of attack on cellodextrins, phosphoric acid-swollen cellulose, and lichenan to give cellobiose, cellotriose, and small proportions of d-glucose. Endo B in addition showed endo-d-xylanase activity.     

Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K_m and 4-fold higher V_max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains.     

Purification and characterization of a new glucose dehydrogenase from vegetative cells of Bacillus megaterium

A new type of glucose dehydrogenase was purified from vegetative cells of Bacillus megaterium IAM1030. The characteristics of the vegetative-cell enzyme were investigated and compared with the enzyme from sporulating cells of B. megaterium IWG3. They are very similar in the following points: molecular size (M_r 120,000), subunit composition (homo tetramer), pH-activity profile with an optimum pH at around 8, pH-stability profile with a stable pH range of 6.0–7.5 (at 25°C, for 30 min), substrate specificity (specific for d-glucose and 2-deoxy-d-glucose), and the affinity for glucose (a K_m value of 11–12 mM at pH 8.0, 2.5 mM NAD). They are a little different in the following points: slower mobility for the vegetative-cell enzyme in polyacrylamide-gel electrophoresis at pH 8, immunological determinants (some of them are common), and higher heat resistance for the vegetative-cell enzyme at pH 6.5. They are quite different in their affinity for NAD and NADP. The K_m values for NAD are 0.1 mM for the vegetative-cell enzyme and 1.0 mM for the spore enzyme, while the values for NADP are 7.1 mM for the vegetative-cell enzyme and 0.09 mM for the spore enzyme, at pH 8.0, 0.1 M d-glucose. These results suggest that B. megaterium has at least two types of glucose dehydrogenase.     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Characterization of d-galactosyl-β1→4-l-rhamnose phosphorylase from Opitutus terrae

We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0–7.0. The values of k_cat and K_m of the phosphorolytic activity of the enzyme on GalRha were 60 s^?1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms.