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Biomedical subjects

H G Floss

Publications and source records attributed to H G Floss.

At least 73 records · Page 4Linked to original sources
PubMed1987

Stereochemistry and mechanism of reactions catalyzed by tyrosine phenol-lyase from Escherichia intermedia.

Stereochemical studies on tyrosine phenol-lyase from Escherichia intermedia have shown that the alpha, beta-elimination reactions of L-serine and D- and L-tyrosine proceed with retention of configuration at C-beta. Stereospecifically beta-tritiated L-serine is slowly racemized at C-beta. Deuterium from the alpha-position of L-tyrosine is partially transferred to C-4 of the phenol formed when the alpha, beta-elimination reaction is carried out in H2O, although no transfer of alpha-1H in 2H2O was seen. The result favors tautomerization of the p-hydroxyphenyl to a cyclohexadienonyl moiety prior to carbon-carbon bond cleavage. In the conversion of L- to D-alanine catalyzed by tyrosine phenol-lyase, some alpha-hydrogen recycling is observed, pointing to a single-base racemization mechanism. Attempts to demonstrate cofactor motion during racemization by NaBH4 reduction of [3H]PLP-enzyme: D- and L-alanine complexes failed, but showed that, as in other PLP enzymes, the holoenzyme is reduced preferentially from the Re face with respect to C-4' of PLP and enzyme-substrate complexes preferentially from the Si face.

Carbon Radioisotopes↗
PubMed1987

Steric course of the rhodium-catalyzed decarbonylation of chiral 4-methyl-[1-3H,2-2H1]pentanal.

(R)- and (S)-4-methyl-[1-3H,2-2H1]pentanal were prepared from L- and D-leucine via leucic acid and (S)- and (R)-4-methyl-[2-2H1]pentanoic acid. Decarbonylation of these samples with tris-(triphenylphosphine)rhodium chloride followed by Kuhn-Roth oxidation of the resulting 2-methylbutane gave chiral acetic acid of 35% e.e. S and 31% e.e. R configuration, respectively. The decarbonylation reaction thus proceeds with net retention of configuration, possibly accompanied by some racemization.

Caproates↗
PubMed1987

Studies on the biosynthesis of the alpha-glucosidase inhibitor acarbose: valienamine, a m-C7N unit not derived from the shikimate pathway.

Feeding experiments with Actinoplanes sp. SN223/29 showed that 3-amino-5-hydroxy-[7-13C]benzoic acid is not incorporated into acarbose (I). The valienamine moiety of I is thus not derived in the same way, from the shikimate pathway, as the m-C7N units in the ansamycin, mitomycin and ansamitocin antibiotics. Feeding experiments with [U-13C3]-glycerol followed by analysis of I by multiple quantum NMR spectroscopy support this conclusion and point to formation of the valienamine moiety by cyclization of a heptulose phosphate which arises from a triose phosphate via successive transfer of two 2-carbon fragments by transketolase, as proposed by Pape and co-workers.

Acarbose↗
PubMed1986

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates.

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.

Candida↗
PubMed1986

Further studies on the biosynthesis of chlorothricin.

Feeding experiments with [U-13C3]- and (2R)-[1-2H2]glycerol showed that glycerol is incorporated intact into carbon atoms 22, 23 and 24 of the aglycone of chlorothricin. C-1 of glycerol gives rise to C-22 with retention of one atom of deuterium, which occupies the H-22R position. A mechanism for the assembly of the aglycone is proposed which invokes phosphoenolpyruvate as the direct precursor of the 3-carbon moiety and a Baeyer-Villiger oxidation as the mode of formation of the macrocyclic lactone functionality. A feeding experiment with [1,2-13C2]succinate suggests that the propionate units of the aglycone polyketide are formed entirely via the methylmalonyl-CoA mutase reaction. The formation of the two 2,6-dideoxy-D-rhamnose moieties of chlorothricin from glucose was shown to involve replacement of the 2-hydroxyl group of the sugar by hydrogen with inversion of configuration at C-2. This contrasts with the retention stereochemistry observed earlier for the analogous formation of the 2,6-dideoxyhexose moiety of the antibiotic granaticin.

Aminoglycosides↗
PubMed1985

Stereochemistry of the kynureninase reaction.

The steric course of the replacement of the anthranilyl group of kynurenine by hydrogen was determined by conversion of (2S,3R)- and (2S,3S)-[3-3H]kynurenine into alanine with kynureninase in D2O, followed by chirality analysis of the alanine methyl group. To minimize enolization, the labeled substrates were generated in situ from the corresponding stereospecifically tritiated tryptophan species. The result, replacement in a retention mode, together with the finding that tritium from the alpha-position of the substrate is recycled and appears both at C alpha and C beta of the product suggests a single base mechanism and an active site geometry of the pyridoxal phosphate-substrate complex in which H alpha and the beta-substituent are syn oriented.

Chemical Phenomena↗
PubMed1985

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.

Bacillus subtilis↗
PubMed1984

Stereochemical course of the transmethylation catalyzed by histamine N-methyltransferase.

The stereochemical course of the methyl group transfer catalyzed by histamine N-methyltransferase was studied using S-adenosylmethionine (AdoMet), which carried a chiral methyl group. The incubation of these AdoMet samples and histamine with a partially purified enzyme obtained from guinea pig brains gave the corresponding methylated histamine. The N-methylhistamine samples were degraded to convert the N-methyl group into the methyl group of acetate using a reaction sequence of known stereochemistry. The results of the configurational analysis of these acetate samples indicated that the enzymatic transfer of the methyl group from the sulfur of AdoMet to the nitrogen of histamine occurs with inversion of configuration.

Animals↗
PubMed1984

Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate.

We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [14C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin.

Candida↗
PubMed1984

Biosynthesis of ketomycin.

The antibiotic ketomycin is formed from shikimic acid via chorismic acid and prephenic acid. Phenylalanine and 2',5'-dihydrophenylalanine are not intermediates in the biosynthesis. Degradation of ketomycin derived from [1,6-14C]shikimic acid showed that prephenic acid is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring, the pro-S-R edge giving rise to the C-2', C-3' side of the cyclohexene ring of ketomycin.

Anti-Bacterial Agents↗
PubMed1983

Biosynthesis of riboflavin. Incorporation of 13C-labeled precursors into the xylene ring.

Growing cultures of Ashbya gossypii were supplemented with various 13C-labeled precursors including [1-13C]acetate, [2-13C]acetate, [1-13C]ribose, [1-13C]glucose, [6-13C]glucose, and [2-13C]glycerol. Riboflavin was isolated from the culture medium, chemically converted to riboflavin tetraacetate, and analyzed by 13C NMR spectroscopy. The xylene ring of riboflavin is formed by dismutation of 6,7-dimethyl-8-ribityllumazine, thus the 8 carbon atoms of the riboflavin xylene ring are composed of 4 biochemically different carbon atoms which are duplicated in the dismutation. The formation of the lumazine from a pyrimidine precursor requires the addition of these 4 carbon atoms which constitute C-6 alpha, C-6, C-7, and C-7 alpha of the lumazine. Results from the present work indicate that these 4 carbon atoms do not arise from acetate, diacetyl, acetoin, or a tetrose, nor from loss of 1 or 2 carbon atoms from either end of a pentose or hexose. Additionally, the data do not support the recent contention that these 4 atoms arise via a dismutation of the pyrimidine precursor of the lumazine. The findings show that there exists a close correspondence between the lumazine carbons 6 alpha, 6, and 7 with C-1, C-2, and C-3, respectively, of a pentose, while C-7 alpha corresponds to C-5 rather than C-4 of a pentose. This in conjunction with results reported earlier indicates an intramolecular rearrangement involving carbons 3, 4, and 5 of a pentose or its biochemical equivalent.

Acetates↗
PubMed1982

Stereochemistry of sodium borohydride reduction of tryptophan synthase of Escherichia coli and its amino acid Schiff's bases.

Tryptophan synthase alpha 2 beta 2 complex containing [4'-3H]pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates. Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate. The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4'. On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced [3H]pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding. Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-[2,3-2H2]serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%). According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3.

Borohydrides↗
PubMed1982

Some stereochemical features of aspartate beta-decarboxylase.

Aspartate beta-decarboxylase catalyzes abortive decarboxylation/transamination of [2-3H]aspartate with at least 17% internal transfer of tritium to the pro-S position at C-4' of the resulting pyridoxamine phosphate. In the normal beta-decarboxylation reaction, at least 1.06% of the tritium from the alpha-position of aspartate appears in the product alanine. The enzyme catalyzes slow hydrogen exchange from the beta-position of alanine but not aspartate. The replacement of the beta-carboxyl group of aspartate by hydrogen occurs in an inversion mode. These results are interpreted in terms of a two-base mechanism.

Alcaligenes↗
PubMed1981

Biosynthesis of the macrolide antibiotic chlorothricin.

Feeding experiments with 13C-labeled precursors followed by 13C NMR analysis of the antibiotic and its aglycon have established a polyketide mode of biosynthesis for chlorothricin, a metabolite of Streptomyces antibioticus. The acyl moiety is a substituted 6-methylsalicylic acid derived from four acetate units and a methyl group of methionine. The aglycon is comprised of ten acetate and two propionate units, leaving three carbon atoms, C-22, -23, and -24, unaccounted for. The two 2-deoxy-D-rhamnose moieties are derived from glucose with retention of the hydrogens at C-1, C-2, and C-6 and loss of H-3 and H-5. The hydrogen at C-4 of glucose is transferred intramolecularly to C-6 of the hexose, replacing the hydroxyl group at C-6 in an inversion mode, a result which implicates the thymidine 5'-diphosphate-glucose oxidoreductase reaction in this transformation.

Acetates↗