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Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

ABSTRACT: Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinckii Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single-crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75?Å. Docking studies identified residues involved in substrate binding, and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggesting that the active site of the enzyme is optimally configured for catalysis with the substrate tested.IMPORTANCE Here we report the crystal structure and biophysical properties of an aldehyde reductase that can detoxify furfural, a common inhibitor of biofuel fermentation found in lignocellulose lysates. The data contained here will serve as a guide for protein engineers to develop improved enzyme variants that would impart furfural resistance to the microorganisms used in biofuel production and thus lead to enhanced biofuel yields from this sustainable resource.

SUBMITTER: Scott AF

PROVIDER: S-EPMC6643236 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Scott Alan F AF Cresser-Brown Joel J Williams Thomas L TL Rizkallah Pierre J PJ Jin Yi Y Luk Louis Y-P LY Allemann Rudolf K RK

Applied and environmental microbiology 20190718 15

Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in <i>Clostridium beijerinckii</i> Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensi ...[more]

PMID: 31101612

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Project description:BackgroundFurfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.ResultsA total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase.ConclusionGenome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.

Project description:Furfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulosic biomass to monomeric sugars, but the molecular response of Clostridium beijerinckii NCIMB 8052 to this compound is unknown. To discern the effect of furfural on C. beijerinckii and to gain insights into the molecular mechanisms of action and detoxification, we studied the physiological changes of furfural-stressed cultures during acetone-butanol-ethanol (ABE) fermentation, and profiled differentially expressed genes by genome-wide transcriptional analysis. C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control. The growth and ABE by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase. By comparing gene expression of furfural-stressed cultures to that of the unstressed control, at both the acidogenic and solventogenic phases, we ascertained that furfural induces expression of several genes including those that code for heat shock proteins, redox enzymes and cofactor associated proteins, and ATP-binding cassette transporters, and represses genes belonging to the phosphotransferase system, two-component system, chemotaxis and cell motility. Based on these results, we discuss the underpinning for furfural-mediated change in ABE fermentation by the solventogenic Clostridium species. C. beijerinckii 8052 pre-culture was incubated anaerobically to attain acidogenic or solventogenic growth phase. The culture was then subdivided into two bottles. One bottle was challenged with furfural and the other bottle was left unchallenged. After 3 h growth, C. beijerinckii 8052 samples were collected, during which the original concentration of furfural in the growth medium was reduced to more than half. Total RNA was isolated, purified, converted to enriched mRNA, and dye-coupled (Alexa Fluor 555) complementary cRNA. This was followed by hybridization and microarray data analysis.

Project description:The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.

Dataset Information

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Publications

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

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