Conversion of (+/-)-synephrine into p-hydroxyphenylacetaldehyde by Arthrobacter synephrinum. A novel enzymic reaction.

A partically purified enzyme from Arthrobacter synephrinum was found to catalyse the conversion of (+/-)-synphrine into p-hydroxyphrenylacetaldehyde and methylamine. The enzyme is highly specific for synephrine and is distinctly different from monoamine oxidase.     

Oxidation of 4-hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A)

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.     

Immunological demonstration of a unique 3,4-dihydroxyphenylacetate 2,3-dioxygenase in soil Arthrobacter strains.

Many bacteria biosynthesize 3,4-dihydroxyphenylacetate 2,3-dioxygenases for growth on aromatic acids, but gram-negative organisms have been most extensively studied. A gram-positive strain containing 2,3-dioxygenase activity was identified as Arthrobacter strain Mn-1. The 2,3-dioxygenase from strain Mn-1 was purified to homogeneity by fast protein liquid chromatography with a Mono Q anion-exchange column. Rabbit polyclonal antidioxygenase antibodies were prepared. Ouchterlony double-diffusion and Western blotting (immunoblotting) protocols were used to probe the distribution of the Mn-1 dioxygenase antigen in soil bacteria. Fourteen 2,3-dioxygenase-containing Bacillus and Pseudomonas strains did not contain immunologically cross-reactive proteins. Six of eight Arthrobacter strains contained 2,3-dioxygenase activity, and all of them produced cross-reactive proteins. The data presented here suggest that a unique type of dioxygenase is geographically widespread but is taxonomically confined to Arthrobacter soil bacteria.     

Immunological demonstration of a unique 3,4-dihydroxyphenylacetate 2,3-dioxygenase in soil Arthrobacter strains.

Many bacteria biosynthesize 3,4-dihydroxyphenylacetate 2,3-dioxygenases for growth on aromatic acids, but gram-negative organisms have been most extensively studied. A gram-positive strain containing 2,3-dioxygenase activity was identified as Arthrobacter strain Mn-1. The 2,3-dioxygenase from strain Mn-1 was purified to homogeneity by fast protein liquid chromatography with a Mono Q anion-exchange column. Rabbit polyclonal antidioxygenase antibodies were prepared. Ouchterlony double-diffusion and Western blotting (immunoblotting) protocols were used to probe the distribution of the Mn-1 dioxygenase antigen in soil bacteria. Fourteen 2,3-dioxygenase-containing Bacillus and Pseudomonas strains did not contain immunologically cross-reactive proteins. Six of eight Arthrobacter strains contained 2,3-dioxygenase activity, and all of them produced cross-reactive proteins. The data presented here suggest that a unique type of dioxygenase is geographically widespread but is taxonomically confined to Arthrobacter soil bacteria.     

Catabolism of 3- and 4-hydroxyphenylacetate by the 3,4-dihydroxyphenylacetate pathway in Escherichia coli.

Various strains of Escherichia coli (but not strain K-12) were found to grow on 3-hydroxyphenylacetate and 4-hydroxyphenylacetate. Both compounds were catabolized by the same pathway, with 3,4-dihydroxyphenylacetate as a substrate for fission of the benzene nucleus, and with pyruvate and succinate as products. All the necessay enzymes were demonstrated in cell extracts prepared from induced cells but were essentially absent from uninduced cells. Mutants unable to grow on 3- and 4-hydroxyphenylactetate were defective in particular enzymes of the pathway. The characteristics of certain mutants indicated that either uptake or hydroxylation of 3- and 4-hydroxyphenylacetate may involve a common protein component. E. coli also grew on 3,4-hydroxyphenylacetate, with induction of the enzyme necessary for its degradation but not those for the uptake-hydroxylation of 3- and 4-hydroxyphenylacetate.     

节杆菌(Arthrobacter sp.)降解阿魏酸的效能

[背景]蓄积在土壤中的阿魏酸类化感自毒物质对农作物生长产生危害,利用有益微生物分解该类物质是一项有效的治理措施.[目的]从自然界土壤分离获得能高效降解阿魏酸的菌株,并评估典型环境因子对降解效能的影响,以期为该菌在阿魏酸类自毒物质降解领域中的应用提供理论依据.[方法]采用一次性投加高浓度化合物的驯化方法分离筛选得到能有效...     

Genotoxic effects of the chlorinated hydroxyfuranones 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-dichloro-5-hydroxy-2[5H]-furanone in Tradescantia micronucleus assays

This is the first report of clastogenic effects of chlorinated hydroxyfuranones (CHFs) in plants. Two byproducts of water chlorination, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-dichloro 5-hydroxy-2[5H]-furanone (MA) induced a dose dependent increase of micronuclei (MN) in pollen mother cells of Tradescantia when doses up to 100 μg MX and 500 μg MA were applied directly to the inflorescences. In contrast, exposure of the stems in aqueous solutions containing up to 1 mg/I MX and 10 mg/I MA did not cause a positive response.     

The role of histidine 200 in MndD, the Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Arthrobacter globiformis CM-2, a site-directed mutagenesis study

The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA). Based on the recent crystal structure of the MndD–DHPA complex, a series of site-directed mutations were made at a conserved second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200 interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II) center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored. Electronic Supplementary Material Supplementary material is available for this article at     

Novel lipase-catalysed enantioselective deacetylation of (+/-)-5-acetoxy-3-(4-fluorophenyl)-2-phenylisoxazolidine

(+/-)-5-Acetoxy-3-(4-fluorophenyl)-2-phenylisoxazolidine has been synthesised by a highly diastereoselective [3+2] cycloaddition reaction between alpha-(4-fluorophenyl)-N-phenylnitrone and vinyl acetate in good yield. Candida rugosa lipase catalyses the deacetylation of this (+/-)-5-acetoxyisoxazolidine in a highly enantioselective fashion in diisopropyl ether containing n-butanol affording (-)-5-acetoxy-3-(4-fluorophenyl)-2-phenylisoxazolidine in 43% yield and >99% ee.     

Synthetic and novel biocatalytic resolution studies on (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins

Eleven (+/-)-5/6/7-acetoxy-4-aryl-3,4-dihydrocoumarins have been synthesised in two steps starting from the coupling of cinnamic acid/substituted cinnamic acid with appropriate phenols, followed by acetylation in 50-83% overall yields. All hydroxy- and acetoxycoumarins were unambiguously identified on the basis of their spectral data. Candida antarctica lipase-catalysed deacetylation of these racemic acetoxydihydrocoumarins in dioxane occurred with moderate enantioselectivity. This is one of the rare examples of resolution using phenolic ester moiety as a remote handle for chiral recognition by a lipase.     

Transformation of 2-aminoacetophenone to (S)-2-amino-1-phenylethanol by Arthrobacter sulfureus

A new isolate of Arthrobacter sulfureus , when incubated at 50 g resting cells (dry cell wt) l(-1) with 50 g glucose l(-1) and 1 g 2-aminoacetophenone l(-1) in 50 mm potassium buffer (pH 7, 4 ml) at 30 degrees C, produced ( S )-2-amino-1-phenylethanol (e.e. >99%) with 75% yield in 6 h.     

Enantioselective resolution of 2-(1-hydroxy-3-butenyl)-5-methylfuran by immobilized lipase

An efficient and convenient strategy for synthesis of enantiomerically pure S-2-(1-hydroxy-3-butenyl)-5-methylfuran was for the first time described utilizing a lipase-mediated asymmetric acylation in organic solvents. Rhizopus arrhizus lipase was chosen as the biocatalyst, and different immobilization methods including sol–gel encapsulation and covalent attachment were adopted to improve its catalytic characteristics. Various influential factors of the reaction were also investigated. Finally, the results showed that the lipase covalently attached onto epoxy resin exhibited the highest enantioselectivity and operational stability. Under optimized reaction conditions, i.e., n-hexane as the solvent, 5/1 (mol/mol) of vinyl acetate to 2-(1-hydroxy-3-butenyl)-5-methylfuran and 30 °C, the ee value of S-1 reached up to above 98% at 52% conversion with an E value of 99.     

Metabolism of DL-(+/-)-phenylalanine by Aspergillus niger.

A fungus capable of degrading DL-phenylalanine was isolated from the soil and identified as Aspergillus niger. It was found to metabolize DL-phenylalanine by a new pathway involving 4-hydroxymandelic acid. D-Amino acid oxidase and L-phenylalanine: 2-oxoglutaric acid aminotransferase initiated the degradation of D- and L-phenylalanine, respectively. Both phenylpyruvate oxidase and phenylpyruvate decarboxylase activities could be demonstrated in the cell-free system. Phenylacetate hydroxylase, which required reduced nicotinamide adenine dinucleotide phosphate, converted phenylacetic acid to 2- and 4-hydroxyphenylacetic acid. Although 4-hydroxyphenylacetate was converted to 4-hydroxymandelate, 2-hydroxyphenylacetate was not utilized until the onset of sporulation. During sporulation, it was converted rapidly into homogentisate and oxidized to ring-cleaved products. 4-Hydroxymandelate was degraded to protocatechuate via 4-hydroxybenzoylformate, 4-hydroxybenzaldehyde, and 4-hydroxybenzoate.     

Oxidation of tyrosine residues in proteins by tyrosinase. Formation of protein-bonded 3,4-dihydroxyphenylalanine and 5-S-cysteinyl-3,4-dihydroxyphenylalanine.

A simple and rapid method was developed for the determination of 3,4-dihydroxyphenylalanine (dopa) and 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-cysteinyldopa) in proteins with the use of high-pressure liquid chromatography. With this method, it is demonstrated that mushroom tyrosinase can catalyse hydroxylation of tyrosine residues in proteins to dopa and subsequent oxidation to dopaquinone residues. The dopaquinone residues in proteins combine with cysteine residues to form 5-S-cysteinyldopa in bovine serum albumin and yeast alcohol dehydrogenase, whereas dopa is the major product in bovine insulin, which lacks cysteine residues.     

Inhibition of retinoic acid metabolising enzymes by 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and related compounds

In a search for inhibitors of all-trans retinoic acid (RA)-metabolising enzymes as potential agents for the treatment of skin conditions and cancer we have examined 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (5). Compound (5) is a moderate inhibitor of RA-metabolising enzymes in mammalian cadaverous tissue microsomes and homogenates as well as RA-induced enzymes in cultured human genital fibroblasts and HaCat cells. Overall (5) was more potent than or equipotent with ketoconazole, a standard inhibitor, in the cadaverous systems but less active towards the RA-induced cell culture systems. Examination of the data suggests that RA-induction generates metabolising enzymes not present in the cadaverous systems, which are more susceptible to inhibition by ketoconazole than (5).     

Inhibition of Retinoic Acid Metabolising Enzymes by 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one and Related Compounds

In a search for inhibitors of all-trans retinoic acid (RA)-metabolising enzymes as potential agents for the treatment of skin conditions and cancer we have examined 2-(4-aminophenylmethyl)-6-hydroxy-3,4-dihydronaphthalen-1(2H)-one (5). Compound (5) is a moderate inhibitor of RA-metabolising enzymes in mammalian cadaverous tissue microsomes and homogenates as well as RA-induced enzymes in cultured human genital fibroblasts and HaCat cells. Overall (5) was more potent than or equipotent with ketoconazole, a standard inhibitor, in the cadaverous systems but less active towards the RA-induced cell culture systems. Examination of the data suggests that RA-induction generates metabolising enzymes not present in the cadaverous systems, which are more susceptible to inhibition by ketoconazole than (5).     

Oxidation of linear terpenes and squalene variants by Arthrobacter sp.

Cells of Arthrobacter sp. that had been isolated from soil were used to study oxidation of some linear terpenes and squalene variants. The cells oxidized geraniol, nerol, and farnesol to the corresponding aldehydes, with partial conversion of the geometrical isomerism of the alpha,beta-double bond. The squalene variant, squalene-2,3-oxide, was cleaved to 9,10-epoxygeranylacetone and geranylacetone. Squalene-2,3-22,23-dioxide was cleaved to 9,10-epoxygeranylacetone. These products were optically active, and their stereochemistry and optical purity were determined.