Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

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Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol. / Kim, Taeho; Stogios, Peter J; Khusnutdinova, Anna N et al.
Yn: Journal of Biological Chemistry, Cyfrol 295, Rhif 2, 10.01.2020, t. 597-609.

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

HarvardHarvard

Kim, T, Stogios, PJ, Khusnutdinova, AN, Nemr, K, Skarina, T, Flick, R, Joo, JC, Mahadevan, R, Savchenko, A & Yakunin, AF 2020, 'Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol', Journal of Biological Chemistry, cyfrol. 295, rhif 2, tt. 597-609. https://doi.org/10.1074/jbc.RA119.011363

APA

Kim, T., Stogios, P. J., Khusnutdinova, A. N., Nemr, K., Skarina, T., Flick, R., Joo, J. C., Mahadevan, R., Savchenko, A., & Yakunin, A. F. (2020). Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol. Journal of Biological Chemistry, 295(2), 597-609. https://doi.org/10.1074/jbc.RA119.011363

CBE

Kim T, Stogios PJ, Khusnutdinova AN, Nemr K, Skarina T, Flick R, Joo JC, Mahadevan R, Savchenko A, Yakunin AF. 2020. Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol. Journal of Biological Chemistry. 295(2):597-609. https://doi.org/10.1074/jbc.RA119.011363

MLA

VancouverVancouver

Kim T, Stogios PJ, Khusnutdinova AN, Nemr K, Skarina T, Flick R et al. Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol. Journal of Biological Chemistry. 2020 Ion 10;295(2):597-609. Epub 2019 Rhag 5. doi: 10.1074/jbc.RA119.011363

Author

Kim, Taeho ; Stogios, Peter J ; Khusnutdinova, Anna N et al. / Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol. Yn: Journal of Biological Chemistry. 2020 ; Cyfrol 295, Rhif 2. tt. 597-609.

RIS

TY - JOUR

T1 - Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

AU - Kim, Taeho

AU - Stogios, Peter J

AU - Khusnutdinova, Anna N

AU - Nemr, Kayla

AU - Skarina, Tatiana

AU - Flick, Robert

AU - Joo, Jeong Chan

AU - Mahadevan, Radhakrishnan

AU - Savchenko, Alexei

AU - Yakunin, Alexander F

N1 - © 2020 Kim et al.

PY - 2020/1/10

Y1 - 2020/1/10

N2 - Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

AB - Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

KW - Aldehyde-Lyases/chemistry

KW - Bacillus/enzymology

KW - Biosynthetic Pathways

KW - Butylene Glycols/metabolism

KW - Catalytic Domain

KW - Crystallography, X-Ray

KW - Escherichia coli/enzymology

KW - Industrial Microbiology

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Phylogeny

KW - Protein Engineering

KW - Thermotoga maritima/enzymology

U2 - 10.1074/jbc.RA119.011363

DO - 10.1074/jbc.RA119.011363

M3 - Article

C2 - 31806708

VL - 295

SP - 597

EP - 609

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -