Friedrich Giffhorn

Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides

Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene (mtlK). A 5.5 kb EcoRI/BglII fragment from R. sphaeroides Si4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a protein of 51,404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of R. sphaeroides Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from Escherichia coli and Enterococcus faecalis, with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The mtlK gene can be used for overexpression of MDH in E. coli in order to obtain sufficient amounts of enzyme for further investigations and applications.

Enzyme evolution in Rhodobacter sphaeroides : selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0.11 h-1 (td = 6.3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M(r)) of the native GDH was 110,000. SDS-PAGE resulted in one single band that represented a polypeptide with a M(r) of 28,000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4.2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.

Goal-oriented screening method for carbohydrate oxidases produced by filamentous fungi

Abstract A rapid and convenient screening method for the in situ detection of microbial carbohydrate oxidases was developed on the basis of a H2O2-dependent, peroxidase-catalyzed chromogenic reaction with ABTS. Thus, out of more than 100,000 colonies, obtained from 300 different soil samples, 166 colonies were identified by the dye formation from the chromogen. The corresponding colonies were isolated, purified, and identified as filamentous fungi. Following growth in liquid cultures, both mycelial extracts and culture supernatants of all isolates were assayed for the occurrence of carbohydrate and polyol oxidase activities, respectively. Both activities were negligible in supernatants, but present in the mycelial extracts of most of the isolates. The specific activities determined with various sugar substrates ranged from 1 to 100 mU mg-1 protein. The specific activities determined with polyol substrates were two orders lower, and therefore were considered to be marginal activities of preexisting carbohydrate oxidases. With slight modifications, this screening method should be generally applicable to the detection of all kinds of oxidases, laccases, phenol oxidases, and peroxidases.

Catabolism of 1,5-Anhydro-d-Fructose in Sinorhizobium morelense S-30.7.5: Discovery, Characterization, and Overexpression of a New 1,5-Anhydro-d-Fructose Reductase and Its Application in Sugar Analysis and Rare Sugar Synthesis

ABSTRACT The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-d-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-d-mannitol (AM) and the further conversion of AM to d-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli. In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti, Brucella suis, and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.

Sorbitol dehydrogenase from Pseudomonas sp.: Purification, characterization and application to quantitative determination of sorbitol

Abstract Sorbitol dehydrogenase ( l -iditol: NAD + oxidoreductase, EC 1.1.1.14) was purified from Pseudomonas sp. to apparent homogeneity by (NH 4 ) 2 SO 4 precipitation, chromatography on Q-Sepharose, affinity chromatography on Procion-blue, and chromatography on hydroxylapatite. The relative molecular mass (M r ) of the native sorbitol dehydrogenase was determined to be 65,800 and its isoelectric point was pH 4.7. SDS-PAGE resulted in one single band representing a polypeptide with a M r of 31,300, indicating that the native enzyme is a dimer. Sorbitol dehydrogenase was specific for NAD and catalysed the interconversion of d -glucitol to d -fructose, and of galactitol to d -tagatose, The pH optimum of substrate oxidation was pH 11.0, and that of substrate reduction was pH 6.7. The determined K M values were for NAD, 0.24 m m ; d -glucitol, 9.1 m m ; galactitol, 3.1 m m ; d -fructose, 175.5 m m ; d -tagatose, 10.0 m m ; and NADH. 0.09 m m . The equilibrium constants for d -glucitol and galactitol interconversion were 3.2 and 1.3 nm, respectively. Due to the favorable kinetic properties of sorbitol dehydrogenase, an assay for substrate transformation was elaborated.

Phototrophic Growth of Rhodopseudomonas gelatinosa on Citrate; Accumulation and Subsequent Utilization of Cleavage Products

Rhodopseudomonas gelatinosa grows on citrate anaerobically in the light. Under aerobic conditions citrate cannot be utilized. Citrate is rapidly fermented by Rps. gelatinosa in the dark, and this fermentation is associated with limited growth (one generation). Anaerobically in the light citrate is decomposed much faster than its catabolic products can be used for the synthesis of cellular material. Therefore, acetate and malate are accumulated in the medium. The acetate concentration in the medium after complete consumption of citrate is higher than the original citrate concentration. Acetate is formed by the citrate lyase reaction and by partial degradation of oxaloacetate. After exhaustion of citrate, growth of Rps. gelatinosa continues at the expence of acetate.

Activation and inactivation of citrate lyase ligase from Rhodopseudomonas gelatinosa

Rhodopseudomonas gelatinosa is one of the few microorganisms capable of growing on citrate anaerobically in the light [ 1,2] . The first enzyme involved in citrate breakdown by this bacterium is citrate lyase (EC 4.1.3.6) which carries acetyl groups in thioester linkage at its active sites. Splitting off the acetyl groups results in the inactive form of the enzyme (deacetyl-citrate lyase) [3,4] . It could be shown in our laboratory that citrate lyase of R. gelatinosa is subject to covalent modification. Upon depletion of citrate in the medium, the lyase is inactivated by a specific deacetylase. Addition of citrate to the medium causes rapid activation of the lyase by acetylation [5,6] . The enzyme responsible for the latter reaction is citrate lyase ligase:

Crystallization and subunit composition of citrate lyase of Rhodopseudomonas gelatinosa.

Previously, a purification procedure for citrate lyase (EC 4.1.3.6) of Rhodopseudomonas gelathosa has been described [l] which because of the instability of the enzyme in some purification steps and of the low yields is not suited for the preparation of large amounts of citrate lyase. In connection with studies on the enzymes which modify citrate lyase by acetylation or deacetylation [2,3] it was necessary to prepare large amounts of enzyme. The procedure used for this purpose is reported here. It involves 2 very effective crystallization steps and yields preparations with a specific activity much higher than measured with purified citrate lyases from other sources. Sodium dodecylsulfate gel electrophorcsis revealed the presence of 3 types of subunits in the enzyme as has been shown for citrate lyase from other microorganisms [4-71.

Isolation and characterization of a virulent phage for Rhodobacter sphaeroides

A new virulent bacteriophage, termed øRsV, was isolated from a local sewage plant on the facultative phototrophic bacterium Rhodobacter sphaeroides DSM 159 as the host organism. Electron microscopic studies revealed that in general morphology phage øRsV resembles the T-even Escherichia coli phages. The host range of phage øRsV was restricted to strains of R. sphaeroides. E. coli strains B and K 12 were not infected. The phage genome was characterized on the basis of thermal denaturation profiles and restriction analyses indicating that it consists of about 160 kb of double-stranded DNA lacking cohesive ends. The G+C content was determined to be 46.8 mol%.