6msw
Crystal structure of BH1352 2-deoxyribose-5-phosphate from Bacillus halodurans, K184L mutant
Structural highlights
FunctionDEOC_HALH5 Catalyzes a reversible aldol reaction between acetaldehyde and D-glyceraldehyde 3-phosphate to generate 2-deoxy-D-ribose 5-phosphate.[HAMAP-Rule:MF_00114] Publication Abstract from PubMedCarbon-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 A resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr(224) in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys(155) 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. Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.,Kim T, Stogios PJ, Khusnutdinova AN, Nemr K, Skarina T, Flick R, Joo JC, Mahadevan R, Savchenko A, Yakunin AF J Biol Chem. 2020 Jan 10;295(2):597-609. doi: 10.1074/jbc.RA119.011363. Epub 2019, Dec 5. PMID:31806708[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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