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Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110.

Lee DH, Lee YJ, Ryu YW, Seo JH - Microb. Cell Fact. (2010)

Bottom Line: ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics.The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER.The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.

Results: The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42 degrees C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.

Conclusions: A novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

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Multiple alignment of the deduced amino acid sequence for the ER gene from C. magnoliae JH110 with other AKRs showing erythrose reduction activity. AKRs are identified by their GenBank accession numbers: Human aldose reductase (ALR, AAB88851), barley ALR1 (CAA88322), S. cerevisiae GCY1 (CAA65512), T. megachiliensis SNG-42 ER1 (BAD90687), T. megachiliensis SNG-42 ER2 (BAD90688), T. megachiliensis SNG-42 ER3 (BAD90689), and C. magnoliae JH110 ER (FJ550210). The tetra-amino acid motif, IPKS, conserved among NADPH-dependent reductases is indicated by a box. Gray-shaded amino acids are conserved in at least six of the seven AKRs shown. Black-shaded amino acids are conserved in all sequences.
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Figure 1: Multiple alignment of the deduced amino acid sequence for the ER gene from C. magnoliae JH110 with other AKRs showing erythrose reduction activity. AKRs are identified by their GenBank accession numbers: Human aldose reductase (ALR, AAB88851), barley ALR1 (CAA88322), S. cerevisiae GCY1 (CAA65512), T. megachiliensis SNG-42 ER1 (BAD90687), T. megachiliensis SNG-42 ER2 (BAD90688), T. megachiliensis SNG-42 ER3 (BAD90689), and C. magnoliae JH110 ER (FJ550210). The tetra-amino acid motif, IPKS, conserved among NADPH-dependent reductases is indicated by a box. Gray-shaded amino acids are conserved in at least six of the seven AKRs shown. Black-shaded amino acids are conserved in all sequences.

Mentions: By utilizing the recently constructed expressed sequence tag (EST) library of C. magnoliae JH110 in our lab (unpublished data), we discovered a clone containing the ER-encoding gene from the randomly sequenced 912 EST clones. To investigate the existence of introns in this EST clone, PCR was performed using the C. magnoliae JH110 genomic DNA as a template. The full-sequenced PCR product had exactly the same sequence of the EST clone, indicating absence of intron. A 1041-bp C. magnoliae JH110 ER (CmER) with 5'- and 3'-untranslated regions was obtained from the C. magnoliae JH110 genomic DNA and it harbored an open reading frame (ORF) of 849 bp with an ATG initiation codon and a TGA termination codon. This gene encodes a polypeptide of 282 amino acid residues with a predicted molecular mass of 31.4 kDa and an isoelectric point of 6.25. The deduced amino acid sequence of the intronless CmER gene was compared with other protein sequences of aldo-keto reductase (AKR) available from the NCBI database using the BLASTP program. The CmER showed a significant homology to the AKR superfamily. The level of identity showed the highest with aldehyde reductase I from Aspergillus fumigatus (XP 754700, 44% identity), and ER1 (BAD90687, 42% identity) and ER2 (BAD90688, 41% identity) from T. megachiliensis SNG-42. In addition, it was also highly similar to hypothetical protein sequence (XP 662433, 43% identity), which was annotated as AKR in the genome sequence of Aspergillus nidulans. The CmER exhibited no similarity to the well-studied xylose reductases among AKR superfamily. The multiple-sequence alignment analysis conducted using aldose reductases with erythrose reduction activity showed two conserved motifs, GYRH and AYSPL, from yeast to human (Fig. 1). The deduced amino acid sequence of the putative CmER gene was used for the construction of a phylogenetic tree with full length amino acid sequences of AKRs from various organisms [19]. In the phylogenetic tree, the CmER was close to the well-characterized glycerol dehydrogenase coded by GCY1 from Saccharomyces cerevisiae, aldehyde reductase from Sporobolomyces salmonicolor, and ERs from T. megachiliensis SNG-42. Furthermore, the CmER formed a family in the AKR clades separated from other known AKR clades. This result indicates that CmER has evolved differently from the other AKR family members (Fig. 2).


Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110.

Lee DH, Lee YJ, Ryu YW, Seo JH - Microb. Cell Fact. (2010)

Multiple alignment of the deduced amino acid sequence for the ER gene from C. magnoliae JH110 with other AKRs showing erythrose reduction activity. AKRs are identified by their GenBank accession numbers: Human aldose reductase (ALR, AAB88851), barley ALR1 (CAA88322), S. cerevisiae GCY1 (CAA65512), T. megachiliensis SNG-42 ER1 (BAD90687), T. megachiliensis SNG-42 ER2 (BAD90688), T. megachiliensis SNG-42 ER3 (BAD90689), and C. magnoliae JH110 ER (FJ550210). The tetra-amino acid motif, IPKS, conserved among NADPH-dependent reductases is indicated by a box. Gray-shaded amino acids are conserved in at least six of the seven AKRs shown. Black-shaded amino acids are conserved in all sequences.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902421&req=5

Figure 1: Multiple alignment of the deduced amino acid sequence for the ER gene from C. magnoliae JH110 with other AKRs showing erythrose reduction activity. AKRs are identified by their GenBank accession numbers: Human aldose reductase (ALR, AAB88851), barley ALR1 (CAA88322), S. cerevisiae GCY1 (CAA65512), T. megachiliensis SNG-42 ER1 (BAD90687), T. megachiliensis SNG-42 ER2 (BAD90688), T. megachiliensis SNG-42 ER3 (BAD90689), and C. magnoliae JH110 ER (FJ550210). The tetra-amino acid motif, IPKS, conserved among NADPH-dependent reductases is indicated by a box. Gray-shaded amino acids are conserved in at least six of the seven AKRs shown. Black-shaded amino acids are conserved in all sequences.
Mentions: By utilizing the recently constructed expressed sequence tag (EST) library of C. magnoliae JH110 in our lab (unpublished data), we discovered a clone containing the ER-encoding gene from the randomly sequenced 912 EST clones. To investigate the existence of introns in this EST clone, PCR was performed using the C. magnoliae JH110 genomic DNA as a template. The full-sequenced PCR product had exactly the same sequence of the EST clone, indicating absence of intron. A 1041-bp C. magnoliae JH110 ER (CmER) with 5'- and 3'-untranslated regions was obtained from the C. magnoliae JH110 genomic DNA and it harbored an open reading frame (ORF) of 849 bp with an ATG initiation codon and a TGA termination codon. This gene encodes a polypeptide of 282 amino acid residues with a predicted molecular mass of 31.4 kDa and an isoelectric point of 6.25. The deduced amino acid sequence of the intronless CmER gene was compared with other protein sequences of aldo-keto reductase (AKR) available from the NCBI database using the BLASTP program. The CmER showed a significant homology to the AKR superfamily. The level of identity showed the highest with aldehyde reductase I from Aspergillus fumigatus (XP 754700, 44% identity), and ER1 (BAD90687, 42% identity) and ER2 (BAD90688, 41% identity) from T. megachiliensis SNG-42. In addition, it was also highly similar to hypothetical protein sequence (XP 662433, 43% identity), which was annotated as AKR in the genome sequence of Aspergillus nidulans. The CmER exhibited no similarity to the well-studied xylose reductases among AKR superfamily. The multiple-sequence alignment analysis conducted using aldose reductases with erythrose reduction activity showed two conserved motifs, GYRH and AYSPL, from yeast to human (Fig. 1). The deduced amino acid sequence of the putative CmER gene was used for the construction of a phylogenetic tree with full length amino acid sequences of AKRs from various organisms [19]. In the phylogenetic tree, the CmER was close to the well-characterized glycerol dehydrogenase coded by GCY1 from Saccharomyces cerevisiae, aldehyde reductase from Sporobolomyces salmonicolor, and ERs from T. megachiliensis SNG-42. Furthermore, the CmER formed a family in the AKR clades separated from other known AKR clades. This result indicates that CmER has evolved differently from the other AKR family members (Fig. 2).

Bottom Line: ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics.The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER.The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT

Background: Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.

Results: The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42 degrees C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.

Conclusions: A novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

Show MeSH
Related in: MedlinePlus