Purification, characterization and structure analysis of NADPH-dependent d-xylose reductases from Candida tropicalis

Yokoyama, Shin-ichiro; Suzuki, Tomohiko; Kawai, Kazuhiro; Horitsu, Hiroyuki; Takamizawa, Kazuhiro

doi:10.1016/0922-338x(95)90606-z

Cited by 77 publications

(48 citation statements)

References 31 publications

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“…The C. magnoliae ER also had a high cysteine content (2.2% mol content), which is typical of ADRs (26). Indeed, the overall amino acid composition of the C. magnoliae ER is quite similar to that of ADRs from human muscle (33), pig lens (7), and Candida tropicalis (55).…”

Section: Purification Of Ermentioning

confidence: 78%

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Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Lee¹,

Kim²,

Ryu

et al. 2003

Appl Environ Microbiol

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Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K m ‫؍‬ 7.9 mM; k cat /K m ‫؍‬ 0.73 mM ؊1 s ؊1 ). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k cat /K m ‫؍‬ 450 mM ؊1 s ؊1 ) than with NADPH (k cat /K m ‫؍‬ 5.5 mM ؊1 s ؊1 ), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu 2؉ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.Erythritol, a four-carbon polyol, is widely distributed in nature (16). Like most other polyols, erythritol is a metabolite or storage compound and is found in seaweed, mushrooms, and fruits. It is a noncaloric, noncariogenic sweetener that is safe for diabetics (35). Erythritol has about 70% of the sweetness of sucrose in a 10% (wt/vol) solution and a very high negative heat capacity, providing a strong cooling effect when dissolved (16). Erythritol can be synthesized from dialdehyde starch by a high-temperature chemical reaction in the presence of a nickel catalyst (39). This process has not been industrialized because of its low efficiency. Erythritol also can be produced by microbial methods that utilize osmophilic yeasts and some bacteria (42, 48), and it has been produced commercially by using a mutant of Aureobasidium that produces erythritol in high yield (44% [wt/wt] glucose) (20). Recently, a high-erythritol-producing yeast strain was isolated from honeycombs and the new isolate was identified as Candida magnoliae KFCC 11023 (44). This strain can produce erythritol in high yield (43% [wt/wt] glucose) when cultured appropriately (42, 44).There are many repor...

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Section: Purification Of Ermentioning

confidence: 78%

“…All AKRs have a high proline content, but ADRs are clearly distinguished from aldehyde reductases (ALRs) by their higher cysteine contents (9 to 10 versus 3 to 4 Cys residues) (55). The amino acid composition of C. magnoliae ER is high in proline (6.1%), which is typical of AKRs (7, 32).…”

Section: Purification Of Ermentioning

confidence: 96%

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Lee¹,

Kim²,

Ryu

et al. 2003

Appl Environ Microbiol

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“…There is conflicting information, however, regarding XR from Candida tropicalis (AKR2B4). One report suggests that it is specific for NADPH [26], whereas others show dual specificity [27,28]. Since a key determinant for NADH specificity appears to be Glu 227 in C. tenuis XR, we examined this position in the other XRs.…”

Section: Discussionmentioning

confidence: 99%

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases

Kavanagh

Klimacek

Nidetzky

et al. 2003

Biochemical Journal

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Xylose reductase (XR; AKR2B5) is an unusual member of aldoketo reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD + to 1.80 Å resolution (where 1 Å = 0.1 nm) with a crystallographic R-factor of 18.3 %. A comparison of the NAD + -and the previously determined NADP + -bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2 -phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn 276 , which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu 227 on a short rigid helix, which makes hydrogen bonds to both the 2 -and 3 -hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3 -endo conformation rather than the 2 -endo conformation seen in the NADP + -bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

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“…Xylose reductase (XR) is commonly found in yeasts and filamentous fungi, often with several isozymes in one species (21,24,33). XR catalyzes the first step of D-xylose metabolism, reducing xylose to xylitol with concomitant NAD(P)H oxidation.…”

mentioning

confidence: 99%

Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from Neurospora crassa

Woodyer¹,

Simurdiak

Donk³

et al. 2005

Appl Environ Microbiol

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Purification, characterization and structure analysis of NADPH-dependent d-xylose reductases from Candida tropicalis

Cited by 77 publications

References 31 publications

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases

Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from Neurospora crassa

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