Formation of 4-keto-D-aldopentoses and 4-pentulosonates (4-keto-D-pentonates) with unidentified membrane-bound enzymes from acetic acid bacteria

In our previous study, a new microbial reaction yielding 4-keto-D-arabonate from 2,5-diketo-D-gluconate was identified with Gluconacetobacter liquefaciens RCTMR 10. It appeared that decarboxylation and dehydrogenation took place together in the reaction. To analyze the nature of the reaction, investigations were done with the membrane fraction of the organism, and 4-keto-D-arabinose was confirmed as the direct precursor of 4-keto-D-arabonate. Two novel membrane-bound enzymes, 2,5-diketo-D-gluconate decarboxylase and 4-keto-D-aldopentose 1-dehydrogenase, were involved in the reaction. Alternatively, D-arabonate was oxidized to 4-keto-D-arabonate by another membrane-bound enzyme, D-arabonate 4-dehydrogenase. More directly, D-arabinose oxidation was examined with growing cells and with the membrane fraction of G. suboxydans IFO 12528. 4-Keto-D-arabinose, the same intermediate as that from 2,5-diketo-D-gluconate, was detected, and it was oxidized to 4-keto-D-arabonate. Likewise, D-ribose was oxidized to 4-keto-D-ribose and then it was oxidized to 4-keto-D-ribonate. In addition to 4-keto-D-aldopentose 1-dehydrogenase, the presence of a novel membrane-bound enzyme, D-aldopentose 4-dehydrogenase, was confirmed in the membrane fraction. The formation of 4-keto-D-aldopentoses and 4-keto-D-pentonates (4-pentulosonates) was finally confirmed as reaction products of four different novel membrane-bound enzymes.     

Production of 3,4-dihydroxyphthalate from phthalate by a membrane-bound two-enzyme system from Rhodococcus erythropolis

 Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe²⁺ and Mn²⁺ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system. Received: 27 April 1994/Received last revision: 19 August 1994/Accepted: 12 September 1994     

Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal

Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD(+) to NADP(+) as a cofactor, although AKRs generally show higher affinities for the latter.     

High-yield 5-keto-d-gluconic acid formation is mediated by soluble and membrane-bound gluconate-5-dehydrogenases of Gluconobacter oxydans

Gluconobacter oxydans DSM 2343 is known to catalyze the oxidation of glucose to gluconic acid, and subsequently, to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA), by membrane-bound and soluble dehydrogenases. In G. oxydans MF1, in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated, formation of the undesired 2-KGA was absent. This mutant strain uniquely accumulates high amounts of 5-KGA in the culture medium. To increase the production rate of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, we equipped G. oxydans MF1 with plasmids allowing the overproduction of the soluble and the membrane-bound 5-KGA-forming enzyme. Whereas the overproduction of the soluble gluconate:NADP 5-oxidoreductase resulted in the accumulation of up to 200 mM 5-KGA, the detected 5-KGA accumulation was even higher when the gene coding for the membrane-bound gluconate-5-dehydrogenase was overexpressed (240 to 295 mM 5-KGA). These results provide a basis for designing a biotransformation process for the conversion of glucose to 5-KGA using the membrane-bound as well as the soluble enzyme system.The corresponding author contributed equally to the first author.     

Enzyme complex structure and location of enzymes in the phthalate degradative pathway in Rhodococcus erythropolis

A. SUEMORI, K. NAKAJIMA, R. KURANE AND Y. NAKAMURA. 1996. Rhodococcus erythropolis strain S1 formed enzymes essential to the degradation of phthalate when grown in phthalate-minimal medium. The reaction responsible for the dihydroxylation of the phthalate-benzene ring was concluded to be catalysed by membrane-associated phthalate 3,4-dioxygenase (PO). Of the other enzymes involved, 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH) and 3,4-dihydroxyphthalate 2-decarboxylase (PC) appeared likely to be membrane-bound, while protocatechuate 3,4-dioxygenase appeared to be present in the cytoplasm. Based on the data, the membrane-bound PO and PH apparently form an enzyme complex, which is associated with the NADH-regenerating system.     

The crystal structure of SDR-type pyridoxal 4-dehydrogenase of Mesorhizobium loti

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B(6) compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD(+)-bound enzyme.     

Membrane-bound glycerol dehydrogenase catalyzes oxidation of D-pentonates to 4-keto-D-pentonates,D-fructose to 5-keto-D-fructose,and D-psicose to 5-keto-D-psicose

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca²⁺. D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.     

Physiological role of glucoside 3-dehydrogenase and cytochrome c551 in the sugar oxidizing system of Flavobacterium saccharophilum

Flavobacterium saccharophilum cytoplasmic membranes contain several cytochromes linked to the respiratory chain. The presence of c-type cytochrome, cytochrome o, and a small amount of a-type cytochrome was proved. Cytochrome c551 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a membrane fraction of F. saccharophilum and its properties determined. Cytochrome c551 possessed absorption peaks at 407 nm in the oxidized form, and at 415, 521, 551 nm in the reduced form. The cytochrome c551 had a molecular weight of 15,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Glucoside 3-dehydrogenase of F. saccharophilum reduced the cytochrome c551 with methyl-alpha-D-glucoside, D-glucose, sucrose, or validoxylamine A. When the purified glucoside 3-dehydrogenase was incubated with methyl-alpha-D-glucoside and purified ferricytochrome c551, methyl-alpha-D-3-ketoglucoside was formed as indicated by GC-MS analysis. The addition of a substrate to the membrane fraction caused an increase in the rate of oxygen uptake and an abrupt reduction in cytochrome c551. The electron transfer in the 3-keto sugar forming system may be as follows: sugars----glucoside 3-dehydrogenase----cytochrome c551----cytochrome oxidase----O2. Thus, the electron acceptor of glucoside 3-dehydrogenase is possibly connected to the membrane-bound cytochrome system.     

The Crystal Structure of SDR-Type Pyridoxal 4-Dehydrogenase of Mesorhizobium loti

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B₆ compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD⁺-bound enzyme.     

Purification and properties of glucoside 3-dehydrogenase from Flavobacterium saccharophilum

A membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270,000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66,000. The enzyme reacted with various artificial electron acceptors such as 2,6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromercuribenzoate. D-Glucose and methyl-alpha- and beta-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.     

Photo-oxygenation of polyhydroxyalkylfurans: rearrangement of O-acetyl derivatives

The photo-oxygenation of ethyl 2-methyl-5-(1,2,3,4-tetra-O-acetyl-d-lyxo-tetritol-1-yl)-3-furoate, ethyl 2-methyl-5-(1,2,3,4-tetra-O-acetyl-d-arabino-tetritol-1-yl)-3-furoate, 3-acetyl-2-methyl-5-(1,2,3,4-tetra-O-acetyl-d-arabino-tetritol-1-yl)furan, and ethyl 5-(1,4-di-O-acetyl-2,3-O-isopropylidene-d-lyxo-tetritol-1-yl)-2-methyl-3-furoate yielded the corresponding 1,4-endo-peroxides (3a–3d as pairs of diastereomers). Each diastereomer of the pairs 3a and 3d was isolated by fractional crystallisation. The rearrangement of the endo-peroxides at room temperature, by dissolution in CDCl₃, yielded the corresponding diepoxides and monoepoxides. The reduction of 3a–3d with methyl sulphide yielded the corresponding γ-diketones, ethyl (E)-2-C-acetyl-5,6,7,8-tetra-O-acetyl-2,3-dideoxy-d-lyxo-oct-2-en-4-ulosonate, ethyl (E)-2-C-acetyl-5,6,7,8-tetra-O-acetyl-2,3-dideoxy-d-arabino-oct-2-en-4-ulosonate, 3-C-acetyl-6,7,8,9-tetra-O-acetyl-1,3,4-trideoxy-d-arabino-non-3-eno-2,5-diulose, and ethyl (E)-2-C-acetyl-5,8-di-O-acetyl-2,3-dideoxy-6,7-O-isopropylidene-d-lyxo-oct-2-en-4-ulosonate, which can isomerise into the corresponding Z isomers.     

Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma.

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.     

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.     

Occurrence in Brain Lysosomes of a Sialidase Active on Ganglioside

A lysosomal preparation, obtained from brain homogenate of 17-day-old C57BL mice by centrifugation on a self-generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40-120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GD1a and fluorimetrically with 4-methylumbelliferyl-1-alpha-D-N-acetylneuraminic acid (MUB-NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane-bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma membrane-bound enzyme. The apparent Km values for GD1a and MUB-NeuAc were 1.5 X 10(-5) and 4.2 X 10(-5) M, respectively, for the lysosomal enzyme and 2.7 X 10(-4) and 6.3 X 10(-5) M for the plasma membrane-bound one. Triton X-100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane-bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane-bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane-bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-1-Phosphate-Negative Mutant of Agrobacterium tumefaciens

Glucose-1-phosphate-negative mutants that are unable to grow in a synthetic medium containing glucose-1-phosphate (G-1-P) as a sole carbon source were isolated by treatment of Agrobacterium tumefaciens IAM 1525 with N-methyl-N'-nitro-N-nitrosoguanidine. All of the enzymes involved in G-1-P metabolism (glucoside-3-dehydrogenase, 3-ketoglucose-1-phosphate-degrading enzyme, alpha-glucosidase, and phosphatases) were detected in the sonic extract prepared from resting cells of one of the mutants, strain M-24, in approximately equal levels to those in the parent strain. Resting cells of the mutant oxidized G-1-P to 3-ketoglucose-1-phosphate (3KG-1-P), the first product in G-1-P metabolism by the bacterium, with little subsequent degradation, whereas the parent showed further degradation of G-1-P via 3KG-1-P. Glucoside-3-dehydrogenase catalyzing 3-ketoglucoside formation was readily released from cells by osmotic shock, whereas the 3KG-1-P-degrading enzyme was not released. Thus, the former and the latter enzymes might be at different intracellular loci, such as periplasm and cytoplasm, respectively. It is suggested that the mutant strain M-24 is a G-1-P-negative mutant deficient in a 3KG-1-P transport system located on the cytoplasmic membrane.     

3-甾酮-1-脱氢酶基因的表达及甾体转化分析

本文利用带有P43启动子的表达分泌载体pWB980,实现了简单节杆菌3-甾酮-1-脱氢酶在枯草芽孢杆菌中的表达,表达出的目的蛋白的分子量为55KDa。利用分光光度法检测得到胞内和胞外可溶性部分的酶活分别为110±0.5mU和15±0.6mU每毫克蛋白, 比出发菌株简单节杆菌提高了将近30倍。重组芽孢杆菌对甾体底物4-AD的转化率为45.3％,比出发菌株简单节杆菌提高了近10倍。利用枯草芽孢杆菌对甾体底物进行脱氢为甾体药物的生产开辟了一个新的途径。     

Solubilization of the chemokine receptor CXCR4

The chemokine receptor CXCR4 was solubilized from the human T-cell line CEM by using the detergent n-dodecyl-beta-maltoside (DDM) and cholesteryl hemisuccinate ester (CHS). Binding studies with (125)I-SDF-1alpha revealed a dissociation constant of 5.33 nM and a receptor density (B(max)) of 2.68 pmol/mg in CEM membranes at 4 degrees C. The affinity of solubilized CXCR4 for SDF-1alpha was identical to membrane-bound CXCR4. Binding of gp120 to solubilized CXCR4 was demonstrated by coprecipitation of gp120 with anti-CXCR4 antibodies.     

Identification, characterization, and partial purification of 4 alpha-carboxysterol-C3-dehydrogenase/ C4-decarboxylase from Zea mays.

A microsomal preparation from seedlings of Zea mays catalyzed the NAD+-dependent oxidative decarboxylation of several substrates, including 4alpha-carboxy-cholest-7-en-3beta-ol, synthesized according to a new procedure, giving the first in vitro evidence for this enzymatic activity in a higher plant. A GC assay has been developed to detect the Delta7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4alpha-Carboxysterol decarboxylation shows an exclusive requirement for an oxidized pyridine nucleotide, with NAD+ being more efficient than NADP+. The decarboxylation reaction is independent of molecular oxygen. 4alpha-Carboxysterol-C3-dehydrogenase/C4-decarboxylase (4alpha-CD) is a microsome-bound protein which can be efficiently solubilized by detergents, including Brij W-1 and sodium cholate. The Brij W-1-solubilized enzyme was partially purified 290-fold by a combination of DEAE anion-exchange chromatography, Cibacron blue 3GA-agarose dye chromatography, and gel permeation. The apparent molecular mass of 4alpha-CD in sodium cholate was estimated to be 45 kDa. These results support the contention that demethylation at C4 of plant sterols is composed of two separate processes: an oxygen- and NAD(P)H-dependent oxidation of the 4alpha-methyl group to produce the 4alpha-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.