Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability

Choi, Jin Kyeong; Lee, Yong Jik; Cao, Thinh Phat; Shin, Sun Mi; Park, Min Kyu; Lee, Han Seung; Luccio, Eric di; Kim, Seong Bo; Lee, Sang Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong Woo

doi:10.1016/j.abb.2016.02.033

Cited by 29 publications

(18 citation statements)

References 45 publications

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“…Most probably, this behavior also correlates with the different specific activities observed for both proteins. Other characterized l -AIs have tetrameric [ 27 ] or hexameric [ 28 , 29 ] structures.…”

Section: Resultsmentioning

confidence: 99%

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

Sousa

Manzo

Garcı́a³

et al. 2017

Molecules

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l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

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Section: Resultsmentioning

confidence: 99%

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

Sousa

Manzo

Garcı́a³

et al. 2017

Molecules

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“…The crystal structure of all the above 3 l‐ AI indicates the presence of hexameric assembly. Understanding of the crystal structures of l‐ AI opens the door for application of protein engineering tools (directed evolution and site directed mutagenesis) to modify the enzyme properties such as substrate specificity, temperature optima, thermostability, pH optima, and enzyme activity pertaining to tagatose production (Choi et al., ). Different strategies of protein engineering have been employed for improving the properties of l‐ AI (Figure ).…”

Section: Approaches For Improvement Of Tagatose Productionmentioning

confidence: 99%

“…The crystal structure of E. coli l‐ arabinose isomerase (ECAI) was first solved by Manjasetty & Chance in . Later on, the crystal structure of l‐ AIs from Lactobacillus fermentum CGMCC2921 (Xu, Li, Liang, Feng, & Xu, ) and Geobacillus kaustophilus (Choi et al., ) were also solved. The crystal structure of all the above 3 l‐ AI indicates the presence of hexameric assembly.…”

Section: Approaches For Improvement Of Tagatose Productionmentioning

confidence: 99%

Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications

Roy

Chikkerur

Roy

et al. 2018

Journal of Food Science

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Nutraceuticals are gaining importance owing to their potential applications in numerous sectors including food and feed industries. Among the emerging nutraceuticals, d‐tagatose occupies a significant niche because of its low calorific value, antidiabetic property and growth promoting effects on beneficial gut bacteria. As d‐tagatose is present in minute quantities in naturally occurring food substances, it is produced mainly by chemical or biological means. Recently, attempts were made for bio‐production of d‐tagatose using l‐arabinose isomerase enzyme to overcome the challenges of chemical process of production. Applications of d‐tagatose for maintaining health and wellbeing are increasing due to growing consumer awareness and apprehension against modern therapeutic agents. This review outlines the current status on d‐tagatose, particularly its production, properties, biological role, applications, and the future perspectives.

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“…To address the kinetic issue, several groups have used enzyme engineering methods to enhance catalytic efficiency of LAI toward galactose and have shown moderate increases in productivity 8–13 . To counter low-stability issues, many groups have tested the utility of thermophilic enzymes 12,14–16 . However, most thermophilic enzymes rely on divalent metal ions (Mn 2+ , Co 2+ , Fe 2+ ) for stability 17 , and high reaction temperatures (≥ 80 °C) result in significant caramelization 18 , which are all undesirable and must be removed from product, adding to processing costs.…”

mentioning

confidence: 99%

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

Bober

Nair

2019

Preprint

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3There are many enzymes that are relevant for making rare and valuable chemicals that 4 while active, are severely limited by thermodynamic, kinetic, or stability issues (e.g. 5 isomerases, lyases, transglycosidase etc.). In this work, we study an enzymatic reaction 6 system − Lactobacillus sakei L-arabinose isomerase (LsLAI) for D-galactose to D-tagatose 7 isomerizationthat is limited by all three reaction parameters. The enzyme has a low 8 catalytic efficiency for non-natural substrate galactose, has low thermal stability at 9 temperatures > 40 °C, and equilibrium conversion < 50%. After exploring several strategies 10 to overcome these limitations, we finally show that encapsulating the enzyme in a gram-11 positive bacterium (Lactobacillus plantarum) that is chemically permeabilized can enable 12 reactions at high rates, high conversion, and at high temperatures. The modified whole cell 13 system stabilizes the enzyme, differentially partitions substrate and product across the 14 membrane to shift the equilibrium toward product formation enables rapid transport of 15 substrate and product for fast kinetics. In a batch process, this system enables 16 approximately 50 % conversion in 4 h starting with 300 mM galactose (an average 17 productivity of 37 mM/h), and 85 % conversion in 48 h, which are the highest reported for 18 food-safe mesophilic tagatose synthesis. We suggest that such an approach may be 19 invaluable for other enzymatic processes that are similarly kinetically-, thermodynamically-20 , and/or stability-limited. 65We use the food-safe engineered probiotic bacterium Lactobacillus plantarum as the 66 expression host due to its increasing relevance to biochemical and biomedical research 30,31 . 67This approach enabled ~ 50 % conversion of galactose to tagatose in 4 h (productivity of ~ 68 38 mmol tagatose L -1 h -1 ) ultimately reaching ~ 85% conversion after 48 h at high galactose 69 loading (300 mM) in batch culture. This is among the highest conversions and 70 productivities reported to date for tagatose production using a mesophilic enzyme. Such an 71 approach is expected to be applicable to other biocatalytic systems where similar trade-offs 72 between kinetics, thermodynamics, and/or stability pose hurdles to process development. 73 74 Results: 75Characterizing L. sakei LAI (LsLAI) limitations. 76 At high substrate loading (400mM galactose) and 37 °C, the reported optimal temperature 77 for this enzyme 32 , LsLAI purified from L. plantarum exhibited an initial forward reaction 78 (turnover) rate (kinitial) of 9.3 ± 0.3 s -1 , which is lower than the maximum reaction rate 79 possible by this enzyme of 17.0 ± 1.3 s -1 min -1 with its preferred substrate, arabinose, at 400 80 mM. Increasing the reaction temperature to 50 °C increased the initial reaction rate to 11.2 81 s -1 ( Fig. 1a) but was accompanied by rapid enzyme inactivation, which is consistent with 82 previous reports of thermal instability of this enzyme 32 . The enzyme exhibited first-order 83 degradation with a half-life (t½) ...

show abstract

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability

Cited by 29 publications

References 45 publications

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications

Surpassing thermodynamic, kinetic, and stability barriers to isomerization catalysis for tagatose biosynthesis

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