Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase from<i>Streptomyces hygroscopicus</i>

Sugimoto, Norihiko; Takakura, Yasuaki; Shiraki, Kentaro; Honda, Shinya; Takaya, Naoki; Hoshino, Takayuki; Nakamura, Akira

doi:10.1271/bbb.130486

Cited by 9 publications

(13 citation statements)

References 37 publications

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“…In fact, it took a combination of natural evolution, DNA shuffling, and random amino acid duplications to confer a similar degree of thermostability to a hygromycin phosphotransferase from Streptomyces hygroscopicus (Hyg10). 12 …”

Section: Resultsmentioning

confidence: 99%

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

Bosch

Sanchez-Freire

Pozo

et al. 2021

ACS Sustainable Chem. Eng.

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The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature ( T m ) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy- l -rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in T m compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a T m 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

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Section: Resultsmentioning

confidence: 99%

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

Bosch

Sanchez-Freire

Pozo

et al. 2021

ACS Sustainable Chem. Eng.

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“…Previously, enzyme kinetics have been reported for both APH(7 00 )-Ia and APH(4)-Ia. The k cat values of APH(7 00 )-Ia and APH(4)-Ia for HygB are almost the same; on the other hand, the K m value of APH(7 00 )-Ia is about ten times greater than that of APH(4)-Ia (Sugimoto et al, 2013;Nakamura et al, 2008). HygB interacted with six residues at the active site in both cases; however, HygB in APH(4)-Ia and APH(7 00 )-Ia forms interactions with nine and five atoms, respectively [ Fig.…”

Section: Figurementioning

confidence: 84%

“…3.4. Thermostable mutants of APH(7 0 0 00 0 0 )-Ia and APH(4)-Ia Thermostable mutants of APH(7 00 )-Ia and APH(4)-Ia have been reported previously, which exhibit optimum temperatures that are 13 and 5 C higher than those of the wild types (Sugimoto et al, 2013;Nakamura et al, 2008). They are denoted HYG10 and HPH5 as ten and five residues are mutated, respectively, in the enzymes, which were obtained by the directed-evolution method.…”

Section: Figurementioning

confidence: 95%

“…The gene encoding APH(7 00 )-Ia (EC 2.7.1.119) from S. hygroscopicus had been cloned into pET-21a(+) vector (Novagen) with a C-terminal 6ÂHis tag as reported previously (Sugimoto et al, 2013). The expression plasmid for APH(7 00 )-Ia was transformed into the E. coli B834(DE3) strain to introduce selenomethionine (SeMet) into the enzyme.…”

Section: Methodsmentioning

confidence: 99%

“…Since APH(4)-Ia and APH(7 00 )-Ia show low homology in amino-acid sequence, we determined the crystal structure of APH(7 00 )-Ia to elucidate the mechanism of phosphorylation of HygB at a different position to APH(4)-Ia. Furthermore, a thermostable mutant of APH(7 00 )-Ia has been obtained which exhibited a 13 C higher optimum temperature than that of the wild type (Sugimoto et al, 2013). Therefore, the crystal structure is also attractive to elucidate the thermostabilization mechanism of the mutant enzyme.…”

Section: Introductionmentioning

confidence: 99%

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Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

et al. 2019

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Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular‐biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7′′‐phosphotransferase‐Ia [APH(7′′)‐Ia] from Streptomyces hygroscopicus and aminoglycoside 4‐phosphotransferase‐Ia [APH(4)‐Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7′′ and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)‐Ia was reported as a ternary complex with HygB and 5′‐adenylyl‐β,γ‐imidodiphosphate (AMP‐PNP). To investigate the differences in the substrate‐recognition mechanism between APH(7′′)‐Ia and APH(4)‐Ia, the crystal structure of APH(7′′)‐Ia complexed with HygB is reported. The overall structure of APH(7′′)‐Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)‐Ia, and consists of an N‐terminal lobe (N‐lobe) and a C‐terminal lobe (C‐lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7′′)‐Ia and APH(4)‐Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C‐lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7′′)‐Ia and APH(4)‐Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.

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Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

Ohta

Hatada

Hidaka

et al. 2014

Appl Microbiol Biotechnol

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A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

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Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase fromStreptomyces hygroscopicus

Cited by 9 publications

References 37 publications

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

Structural basis for the substrate recognition of aminoglycoside 7′′-phosphotransferase-Ia from Streptomyces hygroscopicus

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

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