<i>Thermotoga neapolitana</i>
            Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in
            <i>Escherichia coli</i>

Hess, J. Michael; Tchernajenko, Vladimir; Vieille, Claire; Zeikus, J. G.; Kelly, Robert M.

doi:10.1128/aem.64.7.2357-2360.1998

Cited by 26 publications

(17 citation statements)

References 26 publications

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“…3). TNXI's unusual melting behavior in the presence of Mg 2+ and Co 2+ was noted previously [50]. Further investigations showed that the relative size of the transitions changed when the pH was increased from 7.0 to 7.9 [51], suggesting that the unusual melting behavior was related to metal binding affinity.…”

Section: Discussionsupporting

confidence: 62%

“…Presumably, TNXI does not denature until the all the metals are lost. This pathway, however, would only describe first order inactivation, as the only active species are the tetramer (T) and dimer (D), which have similar biochemical and biophysical properties for TNXI [50]. It is possible that the active intermediate is a form D¢ between D and D*, where only one of the active sites in the dimer is active.…”

Section: Discussionmentioning

confidence: 99%

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Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

et al. 2005

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Enzymes from hyperthermophiles are intrinsically thermostable and thermoactive, with optimal temperatures of activity often in excess of 100°C [1]. Studies focusing on the molecular basis for the thermostability of these enzymes have revealed an array of subtle contributing factors, including larger hydrogen bonding and ion pairing networks, additional inter-subunit interactions in oligomeric proteins, decreased labile amino acid content, lower surface-to-volume ratios, and smaller loops [2][3][4]. These factors can often be identified by comparing the structures of homologous enzymes with varying degrees of thermostability [5]. However, the extent to which these individual factors contribute to thermostability is highly specific to particular enzymes [6].A potential contributing factor to enhanced thermostability that has not been examined in much detail is the role that metals play in stabilizing and activating enzymes from hyperthermophiles in comparison with their less thermophilic counterparts. Xylose isomerase The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co 2+ or Mn 2+ (and Mg 2+ to a lesser extent), the melting temperature [(T m ) %100°C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110°C and 114°C. TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process. TNXI inactivation in the presence of 5 mm metal at 99-100°C was slowest in the presence of Mn 2+ [half-life (t 1 ⁄ 2 ) of 84 min], compared to Co 2+ (t 1 ⁄ 2 of 14 min) and Mg 2+ (t 1 ⁄ 2 of 2 min). While adding Co 2+ to Mg 2+ increased TNXI's t 1 ⁄ 2 at 99-100°C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.Abbreviations TIM, triosephosphate isomerase; XI, xylose isomerase.

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

et al. 2005

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“…These results indicate that BLXI is expressed as a homotetramer in E. coli. It is interesting to note that XIs from the thermophile Thermoanaerobacterium thermosulfurigenes and the hyperthermophile T. neapolitana, both homotetramers in their native forms, are expressed as active dimers in E. coli [38].…”

Section: Purification Of the Recombinant Protein And Physical Propertiesmentioning

confidence: 99%

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

Vieille

Epting

Kelly

et al. 2001

European Journal of Biochemistry

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Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile aminoacid residues (i.e. N and Q) in hyperthermophilic proteins. The N þ 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 þ 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 8C, which is significantly higher than the optimal growth temperature (37 8C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 8C 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 , Mg 2þ -enzyme , Co 2þ -enzyme < Mn 2þ -enzyme, with melting temperatures of 50.3 8C, 53.3 8C, 73.4 8C, and 73.6 8C. 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·mol 21 (apoenzyme) to 604 kJ·mol 21 (Mg 2þ -enzyme) to 1166 kJ·mol 21 (Co 2þ -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 þ 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 a-amylase from B. licheniformis was found to have an average N þ 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.Keywords: Bacillus licheniformis; metal binding; thermostability; xylose isomerase.It has become apparent that protein thermostability arises not from a single chemical or physical factor, but from numerous subtle contributions integrated over the entire molecular structure [1 -6]. Thermostable proteins usually exhibit no significant differences in backbone conformation when compared with less thermostable proteins, but they typically have increased numbers of salt bridges, side chain-side chain hydrogen bonds, and residues involved in a helices [7 -9]. Stability at very high temperatures further requires that a particular enzyme resist thermally induced deleterious chemical reactions, which usually occur at insignificant rates at lower temperatures [10]. For example, one of the mos...

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“…Both GIs are thermostable and active above 90°C (Starnes et al, 1993a(Starnes et al, , 1993bZeikus et al, 1998). Aside from their signi®cantly higher thermostabilities, these two enzymes share many structural and functional characteristics with class II GIs from less thermophilic organisms (Hess and Kelly, 1999;Hess et al, 1998;Sriprapundh et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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

Bandlish

Hess

Epting

et al. 2002

Biotech & Bioengineering

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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.

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Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

Cited by 26 publications

References 26 publications

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

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

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

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