The diversity of microbial aldo/keto reductases from Escherichia coli K12

Lapthorn, A.J.; Zhu, Xiaofeng; Ellis, Elizabeth M.

doi:10.1016/j.cbi.2012.10.008

Cited by 16 publications

(15 citation statements)

References 51 publications

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“…A total of nine open reading frames that encode AKRs in E. coli have been identified using sequence similarity searches. 48 Unlike many other E. coli…”

Section: Discussionmentioning

confidence: 99%

Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli

2014

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Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.

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“…A total of nine open reading frames that encode AKRs in E. coli have been identified using sequence similarity searches. 48 Unlike many other E. coli…”

Section: Discussionmentioning

confidence: 99%

Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli

2014

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“…The difference between the STM2406 gel filtration results and PISA prediction may be due to the tight monomer packing in the oligomeric protein. Experimentally characterized AKRs are mainly monomeric proteins, but there are also examples of dimeric, trimeric, and octameric enzymes (17,23).…”

Section: Resultsmentioning

confidence: 99%

“…data on the functional assignment and characterization of microbial AKRs compared with that for mammalian enzymes (23). Presently, there are Ͼ190 experimentally annotated proteins in the AKR Superfamily Homepage (http://www.med.upenn.edu/ akr/), which fall into 16 families with different substrate preferences (24).…”

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confidence: 99%

“…To date, several biotechnological applications of AKRs have been reported, including the synthesis of drugs, fuels, and intermediates for the production of fine chemicals (31)(32)(33). However, there is a paucity of data on the biochemical and structural characterizations of microbial AKRs compared with those of the mammalian enzymes (23). In this study, we performed biochemical and structural characterizations of microbial AKRs with the aim of identifying efficient reductases for the last step of the DERA-AKR pathway (reduction of 3-HB to 1,3-BDO; Fig.…”

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confidence: 99%

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Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

Kim

Flick

Brunzelle

et al. 2017

Appl Environ Microbiol

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The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in k cat /K m . Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.KEYWORDS aldo-keto reductase, 1,3-butanediol, crystal structure, site-directed mutagenesis, biocatalysis, biotechnology P resently, most of the large-volume chemicals and polymer building blocks used by the chemical industry are manufactured from nonrenewable feedstocks, such as oil and natural gas. Each year, over 80 million tons of industrial chemicals are produced

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“…AKR2E5 showed similarity to most AKRs, although dimers [17,18], trimers (unpublished, PDB ID: 1OG6), tetramers [14], and octamers [19] have also been observed. The overall AKR2E5 structure consisted of a TIM-barrel in the central part of the enzyme, which was also similar to other AKR superfamily members.…”

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confidence: 95%

Structural characterization of an aldo-keto reductase (AKR2E5) from the silkworm Bombyx mori

Yamamoto

Higashiura

Suzuki

et al. 2016

Biochemical and Biophysical Research Communications

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We report a new member of the aldo-keto reductase (AKR) superfamily in the silkworm Bombyx mori. Based on its amino acid sequence, the new enzyme belongs to the AKR2 family and was previously assigned the systematic name AKR2E5. In the present study, recombinant AKR2E5 was expressed, purified to homogeneity, and characterized. The X-ray crystal structures were determined at 2.2 Å for the apoenzyme and at 2.3 Å resolution for the NADPH-AKR2E5 complex. Our results demonstrate that AKR2E5 is a 40-kDa monomer and includes the TIM- or (β/α)8-barrel typical for other AKRs. We found that AKR2E5 uses NADPH as a cosubstrate to reduce carbonyl compounds such as DL-glyceraldehyde, xylose, 3-hydroxy benzaldehyde, 17α-hydroxy progesterone, 11-hexadecenal, and bombykal. No NADH-dependent activity was detected. Site-directed mutagenesis of AKR2E5 indicates that amino acid residues Asp70, Tyr75, Lys104, and His137 contribute to catalytic activity, which is consistent with the data on other AKRs. To the best of our knowledge, AKR2E5 is only the second AKR characterized in silkworm. Our data should contribute to further understanding of the functional activity of insect AKRs.

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The diversity of microbial aldo/keto reductases from Escherichia coli K12

Cited by 16 publications

References 51 publications

Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli

Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

Structural characterization of an aldo-keto reductase (AKR2E5) from the silkworm Bombyx mori

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