Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase

Schauder, Stephan; Schneider, Karl-Heinz; Giffhorn, Friedrich

doi:10.1099/13500872-141-8-1857

Cited by 30 publications

(53 citation statements)

References 34 publications

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“…The putative smoS ribosome binding site (AAGGCGCT) is homologous to the 3Ј end of the R. sphaeroides 16S RNA (13). The smoS ORF encodes a protein consisting of 256 amino acid residues with a predicted molecular mass of 27,012 Da, which is in accordance with the molecular mass (29,000 Da) determined for one subunit of purified SDH (36). The deduced N-terminal amino acid sequence of smoS is consistent with that of the N terminus of SDH as determined by automated Edman degradation (36).…”

Section: Resultssupporting

confidence: 53%

“…The smoS ORF encodes a protein consisting of 256 amino acid residues with a predicted molecular mass of 27,012 Da, which is in accordance with the molecular mass (29,000 Da) determined for one subunit of purified SDH (36). The deduced N-terminal amino acid sequence of smoS is consistent with that of the N terminus of SDH as determined by automated Edman degradation (36). A database search revealed significant homologies between the deduced amino acid sequences of smoS and proteins of the SDR family (21).…”

Section: Resultsmentioning

confidence: 76%

“…A database search revealed significant homologies between the deduced amino acid sequences of smoS and proteins of the SDR family (21). In this protein family are included a large number of highly different enzymes, most of which have homodimeric structures like SDH and do not require metals for catalytic activity (21,36). SDH has the highest degree of identity (34%) with N-acyl-D-mannosamine dehydrogenase from Flavobacte- rium sp.…”

Section: Resultsmentioning

confidence: 99%

“…The mutant strain R. sphaeroides M22 was unable to grow on mannitol and to produce MDH but displayed substantial levels of SDH activity, which originally was assumed to be a marginal activity of MDH. On the basis of the biochemical properties of the isolated enzyme and the homology of the N-terminal amino acid sequence, SDH was preliminarily assigned to the SDR enzymes (36). Southern hybridizations (not shown) of an oligonucleotide probe derived from the N-terminal amino acid sequence of SDH (36) with the 5.5-kb EcoRI-BglII fragment from R. sphaeroides Si4 containing mtlK (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of either substrate, R. sphaeroides Si4 concomitantly induces two NAD ϩ -dependent enzymes: mannitol dehydrogenase (MDH; EC 1.1.1.67 [38]) and sorbitol dehydrogenase (SDH; EC 1.1.1.14 [36]). SDH and MDH are involved in R. sphaeroides Si4 D-glucitol metabolism, but due to its low K m for D-glucitol, SDH is the predominant enzyme for the metabolization of this substrate at low concentrations (36). Both enzymes were purified and characterized.…”

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confidence: 99%

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Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

1997

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The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(؉) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.The purple, nonsulfur bacterium Rhodobacter sphaeroides Si4 is capable of growing on a variety of sugar alcohols (polyols), including D-mannitol and D-glucitol (sorbitol). In the presence of either substrate, R. sphaeroides Si4 concomitantly induces two NAD ϩ -dependent enzymes: mannitol dehydrogenase (MDH; EC 1.1.1.67 [38]) and sorbitol dehydrogenase (SDH; EC 1.1.1.14 [36]). SDH and MDH are involved in R. sphaeroides Si4 D-glucitol metabolism, but due to its low K m for D-glucitol, SDH is the predominant enzyme for the metabolization of this substrate at low concentrations (36). Both enzymes were purified and characterized. SDH is a dimeric enzyme with a subunit molecular mass of 29,000 Da and is active on sorbitol and galactitol, with sorbitol being the preferred substrate. MDH is a monomeric enzyme with a subunit molecular mass of 51,404 Da and is active on D-mannitol, sorbitol, and D-arabitol, with a preference for D-mannitol. Because of the potential of MDH for biotechnological applications (37,38,41), the corresponding gene (mtlK) has been cloned, sequenced (39), and overexpressed (35).In this communication, we demonstrate that the smoS gene, which encodes SDH, is located within a previously unrecognized polyol operon. The gene coding for an ATP-binding protein of an ATP-binding cassette (ABC) transport system (smoK) and the structural genes smoS and mtlK have been identified. On the basis of sequence homology data, SDH can be attributed to the short-chain dehydrogenase/reductase (SDR) protein family (21). Finally, overexpression of SDH in Escherichia coli provides sufficient amounts of the recombinant enzyme for crystallization and structure analysis studies. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains used are listed in Table 1. R. sphaeroides Si4 is an isolate of this laboratory (31). E. coli XL1-Blue MRF...

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Section: Resultssupporting

confidence: 53%

Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

1997

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Asymmetric Synthesis with Recombinant Whole‐Cell Catalysts

2016

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c h a p t e r 2 3 INTRODUCTIONAsymmetric catalytic transformations play a central role in the field of organic chemistry and are of great interest for applications in the chemical and pharmaceutical industry. Among those, hydrolytic and reverse reactions to form carboxylate deriva tives as well as C─C bond-forming and redox reactions are of particular importance. For a long time this field was dominated by the use of chemocatalysts [1]. For example, numerous chiral heavy-metal complexes have been found to be highly efficient cat alysts, on a technical scale, in particular, for the asymmetric reduction of imines, ketones, and enamides. A representative success story is imine reduction for the man ufacture of metolachlor as one of the largest industrially applied asymmetric chemo catalytic processes to date, and the widely technically applied hydrogenation technology for the reduction of ketones and enamides, for which Noyori and Knowles were awarded the Nobel Prize [2]. An alternative for the production of chiral building blocks, with a continuously increasing number of examples, also on an industrial scale, is biocatalysis. Known for a long time, the use of biocatalytic alternatives has also been limited for a long time, which has been due to-among others-the following two reasons: (i) wild-type whole cells such as baker's yeast require a large amount of biomass (due to the forma tion of the desired enzymes in the cell only in low amounts) and often give unsatisfy ing enantioselectivities (due to the action of more than one enzyme), and (ii) the use of isolated enzymes requires additional costs for cell disruption and enzyme isolation as well as the addition of an external amount of cofactor. Due to impressive progress in the field of molecular biology, these limitations have often been overcome. High overexpression of the desired enzyme in recombinant host organism and high cell density fermentation technology provide efficient and economically attractive access to these microorganisms (so-called designer cells), which contain exclusively the desired biocatalyst (enzyme) in large amounts. Such designer cells can be used directly in organic synthetic reactions without the need to isolate the enzyme in additional downstream operation steps. The presence of the cofactor in the cells requires no addition of an external amount of cofactors or supply thereof in very low amount only. Due to these advantages, recombinant whole cells overexpressing cofactor dependent enzymes have become very attractive catalysts for asymmetric synthesis. This is underlined by an increasing number of recent organic biotransformations based on the use of such "designer cells."

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Autodisplay of Active Sorbitol Dehydrogenase (SDH) Yields a Whole Cell Biocatalyst for the Synthesis of Rare Sugars

Jose

Schwichow

2004

ChemBioChem

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Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

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Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase

Cited by 30 publications

References 34 publications

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4

Asymmetric Synthesis with Recombinant Whole‐Cell Catalysts

Autodisplay of Active Sorbitol Dehydrogenase (SDH) Yields a Whole Cell Biocatalyst for the Synthesis of Rare Sugars

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