monica hughes | Newcastle University (original) (raw)

Papers by monica hughes

Archives of Biochemistry and Biophysics, Jun 1, 1994

ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.37) was purified to... more ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.37) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.

Biotechnology & Genetic Engineering Reviews, Nov 1, 1990

Biochemical Journal, Feb 15, 1996

The coding sequence of the mature cyanogenic β--glucosidase (β--glucoside glucohydrolase, EC 3.... more The coding sequence of the mature cyanogenic β--glucosidase (β--glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion β-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-β-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (K m) and the ratio between the maximal velocities and efficiency constants (V max. , V max. \K m) for the hydrolysis of the natural substrate, linamarin, and pnitrophenyl β--glucopyranoside (PNP-Glc) by the recombinant

Plant Molecular Biology, Aug 1, 1991

The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA cl... more The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic beta-glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other beta-glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic beta-glucosidase activity. Extensive homology was found between the white clover beta-glucosidase sequences and a group of prokaryote and mammalian beta-glycosidases. This group of sequences has no homology with a separate set of beta-glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.

Biotechnology and Bioengineering, Feb 5, 1997

Abstract The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta C... more Abstract The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid ...

The Journal of Agricultural Science, Feb 1, 1997

Annals of Botany, Dec 1, 1993

I Cassava (A4uiiihot esculenru Crantz) cDNA clones were used to detect restriction fragment lengt... more I Cassava (A4uiiihot esculenru Crantz) cDNA clones were used to detect restriction fragment length polymorphisms in a collection of Manihr germplasm maintained as in vitro plants at ORSTOM, Montpellier. The collection consisted of mostly African cultivars of M. esculerifu, together with a few A4. gluziovii Mueller von Argau and M. cuerulescens Pohl, and some interspecific hybrids between M. esculerztu and M. gluziovii. The clones revealed significant levels of polymorphism both within and between the species; sufficient to construct dendrograms indicating the genetic diversity within the collection.

beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-b... more beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-beta-D-glucosides as well as glucosides with only a carbohydrate moiety (such as cellobiose). The cyanogenic beta-glucosidase from white clover (subsequently referred to as CBG) is responsible for the cleavage of cyanoglucosides. Both CBG and the cyanoglucosides occur within the plant cell wall where they are found in separate compartments and only come into contact when the leaf tissue experiences mechanical damage. This results in the eventual production of hydrogen cyanide which acts as a deterrent to grazing animals. beta-glucosidases have been assigned to particular glycosyl hydrolase families on the basis of sequence similarity; this classification has placed CBG in family 1 (there are a total of over 40 families) for which a three-dimensional structure has so far not been determined. This is the first report of the three-dimensional structure of a glycosyl hydrolase from family 1. The crystal structure of CBG has been determined using multiple isomorphous replacement. The final model has been refined at 2.15 A resolution to an R factor of 18.9%. The overall fold of the molecule is a (beta/alpha)8 [or (alpha/beta)8] barrel (in common with a number of glycosyl hydrolases) with all residues located in a single domain. Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.

Journal of Molecular Biology, Feb 1, 1993

The cyanogenic beta-glucosidase from Trifolium repens (white clover) has been crystallized, in a ... more The cyanogenic beta-glucosidase from Trifolium repens (white clover) has been crystallized, in a form suitable for X-ray analysis, from ammonium sulphate solutions. The crystals, which diffract to 3.0 A, are tetragonal, space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2. The cell dimensions are a = b = 69.92 A, c = 248.38 A.

Biochemical Journal, Jan 8, 2001

The cDNA encoding ornithine decarboxylase (ODC ; EC 4.1.1.17), a key enzyme in putrescine and pol... more The cDNA encoding ornithine decarboxylase (ODC ; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank2 AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90 % identity with Datura stramonium ODC, and 44 % identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M r of 92 000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both -ornithine and -lysine with K m values of 562 µM and 1592 µM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (K i 1.15 µM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine

Journal of Experimental Botany, 1968

Variation observed in the growth and cytology of white clover cells is con sidered in relation to... more Variation observed in the growth and cytology of white clover cells is con sidered in relation to enzyme production in cultured tissue. Variation also occurred in the production of /3-glucosidase by groups of cultured cells derived from the same plant and with identical cultural histories. This variability between cultures grown in a completely defined medium, provided an approach to the investigation of the factors affecting /3-glucosidase production in cultured cells. Evidence was obtained from Michaelis constants that two distinct /3-glucosidases were produced by cultured tissue.

Phytochemistry, Dec 1, 1971

Abstract Linamarin and lotaustralin were isolated as a mixture from Trifolium repens L. and used ... more Abstract Linamarin and lotaustralin were isolated as a mixture from Trifolium repens L. and used as substrate for the assay of linamarase

Acs Symposium Series, Jul 27, 1993

Biochemical Genetics, 1973

Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by ... more Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by the Li locus in white clover. During purification of enzyme from LiLi plants, there was no separation of activities against the ß-glucosides, p-nitrophenyl ß-d-glucoside, salicin, and linamarin-lotaustralin, and the ß-galactoside, p-nitrophenyl ß-d-galactoside. In addition, tests on mixtures of these four substrates provided no evidence for the existence of more than one enzyme. Immunological tests have shown that plants homozygous for the recessive li allele do not contain an enzymatically inactive protein, antigenically related to the normal enzyme. This suggests that li alleles either specify a low-activity immunologically altered protein or control the synthesis of very low levels of normal enzyme.

Plant Science Letters, Oct 1, 1976

Evidence is presented that ~-glucosidase activity in white clover cells suspended in a liquid med... more Evidence is presented that ~-glucosidase activity in white clover cells suspended in a liquid medium containing glucose, is lower than the ~-glucosidase activity of cells from a non-sugar medium. This difference is not due to differences in the extraction of enzyme activity. The production of ~-glucosidase in cultured cells is demonstrated in a number of genetically different clones of tissue and is not dependent on the presence of functional alleles at the Li locus.

Biochemical Genetics, 1973

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to... more Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl ß-d-glucoside, linamarin-lotaustralin, and p-nitrophenyl ß-d-galactoside. These three

Euphytica, Sep 1, 1982

The Ac allele for cyanoglucoside production in white clover has been shown to be incompletely dom... more The Ac allele for cyanoglucoside production in white clover has been shown to be incompletely dominant and a dosage effect is indicated at this locus. The recessive allele, ac, when homozygous gives rise to absence of cyanoglucosides in the leaf tissue, whereas in heterozygous plants it has been shown to reduce the level of cyanoglucoside, to a level less than

I. PART ONE: Plant genome structure. 1 Nuclear genome. 2. The inheritance of nuclear genes. 3. Ch... more I. PART ONE: Plant genome structure. 1 Nuclear genome. 2. The inheritance of nuclear genes. 3. Chloroplast genome. 4. Mitochondria. 5. Transposable elements. II. PART TWO: Agrobacterium tumefaciens. 6. Agrobacterium tumefaciens. 7. Ti Plasmid as a vector for plant transformation. III. PART THREE: Plant molecular biology. 8. Symbiotic nitrogen fixation. 9. Tissue-specific expression of plant genes: seed storage protein genes. 10. Effect of light on plant development. 11. Flowering. 12. Breeding systems. 13. Reaction of plant to changes in the environment. 14. The molecular biology of disease and pest resistance . IV PART FOUR: An introduction to plant biotechnology. 15. Transgenic plants. 16. Prospects for plant biotechnology.

Archives of Biochemistry and Biophysics, Jun 1, 1994

ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.37) was purified to... more ABSTRACT alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.37) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.

Biotechnology & Genetic Engineering Reviews, Nov 1, 1990

Biochemical Journal, Feb 15, 1996

The coding sequence of the mature cyanogenic β--glucosidase (β--glucoside glucohydrolase, EC 3.... more The coding sequence of the mature cyanogenic β--glucosidase (β--glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion β-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-β-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (K m) and the ratio between the maximal velocities and efficiency constants (V max. , V max. \K m) for the hydrolysis of the natural substrate, linamarin, and pnitrophenyl β--glucopyranoside (PNP-Glc) by the recombinant

Plant Molecular Biology, Aug 1, 1991

The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA cl... more The nucleotide sequence and derived amino acid sequence of two different beta-glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic beta-glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other beta-glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic beta-glucosidase activity. Extensive homology was found between the white clover beta-glucosidase sequences and a group of prokaryote and mammalian beta-glycosidases. This group of sequences has no homology with a separate set of beta-glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.

Biotechnology and Bioengineering, Feb 5, 1997

Abstract The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta C... more Abstract The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid ...

The Journal of Agricultural Science, Feb 1, 1997

Annals of Botany, Dec 1, 1993

I Cassava (A4uiiihot esculenru Crantz) cDNA clones were used to detect restriction fragment lengt... more I Cassava (A4uiiihot esculenru Crantz) cDNA clones were used to detect restriction fragment length polymorphisms in a collection of Manihr germplasm maintained as in vitro plants at ORSTOM, Montpellier. The collection consisted of mostly African cultivars of M. esculerifu, together with a few A4. gluziovii Mueller von Argau and M. cuerulescens Pohl, and some interspecific hybrids between M. esculerztu and M. gluziovii. The clones revealed significant levels of polymorphism both within and between the species; sufficient to construct dendrograms indicating the genetic diversity within the collection.

beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-b... more beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-beta-D-glucosides as well as glucosides with only a carbohydrate moiety (such as cellobiose). The cyanogenic beta-glucosidase from white clover (subsequently referred to as CBG) is responsible for the cleavage of cyanoglucosides. Both CBG and the cyanoglucosides occur within the plant cell wall where they are found in separate compartments and only come into contact when the leaf tissue experiences mechanical damage. This results in the eventual production of hydrogen cyanide which acts as a deterrent to grazing animals. beta-glucosidases have been assigned to particular glycosyl hydrolase families on the basis of sequence similarity; this classification has placed CBG in family 1 (there are a total of over 40 families) for which a three-dimensional structure has so far not been determined. This is the first report of the three-dimensional structure of a glycosyl hydrolase from family 1. The crystal structure of CBG has been determined using multiple isomorphous replacement. The final model has been refined at 2.15 A resolution to an R factor of 18.9%. The overall fold of the molecule is a (beta/alpha)8 [or (alpha/beta)8] barrel (in common with a number of glycosyl hydrolases) with all residues located in a single domain. Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.

Journal of Molecular Biology, Feb 1, 1993

The cyanogenic beta-glucosidase from Trifolium repens (white clover) has been crystallized, in a ... more The cyanogenic beta-glucosidase from Trifolium repens (white clover) has been crystallized, in a form suitable for X-ray analysis, from ammonium sulphate solutions. The crystals, which diffract to 3.0 A, are tetragonal, space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2. The cell dimensions are a = b = 69.92 A, c = 248.38 A.

Biochemical Journal, Jan 8, 2001

The cDNA encoding ornithine decarboxylase (ODC ; EC 4.1.1.17), a key enzyme in putrescine and pol... more The cDNA encoding ornithine decarboxylase (ODC ; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank2 AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90 % identity with Datura stramonium ODC, and 44 % identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M r of 92 000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both -ornithine and -lysine with K m values of 562 µM and 1592 µM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (K i 1.15 µM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine

Journal of Experimental Botany, 1968

Variation observed in the growth and cytology of white clover cells is con sidered in relation to... more Variation observed in the growth and cytology of white clover cells is con sidered in relation to enzyme production in cultured tissue. Variation also occurred in the production of /3-glucosidase by groups of cultured cells derived from the same plant and with identical cultural histories. This variability between cultures grown in a completely defined medium, provided an approach to the investigation of the factors affecting /3-glucosidase production in cultured cells. Evidence was obtained from Michaelis constants that two distinct /3-glucosidases were produced by cultured tissue.

Phytochemistry, Dec 1, 1971

Abstract Linamarin and lotaustralin were isolated as a mixture from Trifolium repens L. and used ... more Abstract Linamarin and lotaustralin were isolated as a mixture from Trifolium repens L. and used as substrate for the assay of linamarase

Acs Symposium Series, Jul 27, 1993

Biochemical Genetics, 1973

Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by ... more Evidence has been sought on the possible existence of multiple forms of the enzyme controlled by the Li locus in white clover. During purification of enzyme from LiLi plants, there was no separation of activities against the ß-glucosides, p-nitrophenyl ß-d-glucoside, salicin, and linamarin-lotaustralin, and the ß-galactoside, p-nitrophenyl ß-d-galactoside. In addition, tests on mixtures of these four substrates provided no evidence for the existence of more than one enzyme. Immunological tests have shown that plants homozygous for the recessive li allele do not contain an enzymatically inactive protein, antigenically related to the normal enzyme. This suggests that li alleles either specify a low-activity immunologically altered protein or control the synthesis of very low levels of normal enzyme.

Plant Science Letters, Oct 1, 1976

Evidence is presented that ~-glucosidase activity in white clover cells suspended in a liquid med... more Evidence is presented that ~-glucosidase activity in white clover cells suspended in a liquid medium containing glucose, is lower than the ~-glucosidase activity of cells from a non-sugar medium. This difference is not due to differences in the extraction of enzyme activity. The production of ~-glucosidase in cultured cells is demonstrated in a number of genetically different clones of tissue and is not dependent on the presence of functional alleles at the Li locus.

Biochemical Genetics, 1973

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to... more Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl ß-d-glucoside, linamarin-lotaustralin, and p-nitrophenyl ß-d-galactoside. These three

Euphytica, Sep 1, 1982

The Ac allele for cyanoglucoside production in white clover has been shown to be incompletely dom... more The Ac allele for cyanoglucoside production in white clover has been shown to be incompletely dominant and a dosage effect is indicated at this locus. The recessive allele, ac, when homozygous gives rise to absence of cyanoglucosides in the leaf tissue, whereas in heterozygous plants it has been shown to reduce the level of cyanoglucoside, to a level less than

I. PART ONE: Plant genome structure. 1 Nuclear genome. 2. The inheritance of nuclear genes. 3. Ch... more I. PART ONE: Plant genome structure. 1 Nuclear genome. 2. The inheritance of nuclear genes. 3. Chloroplast genome. 4. Mitochondria. 5. Transposable elements. II. PART TWO: Agrobacterium tumefaciens. 6. Agrobacterium tumefaciens. 7. Ti Plasmid as a vector for plant transformation. III. PART THREE: Plant molecular biology. 8. Symbiotic nitrogen fixation. 9. Tissue-specific expression of plant genes: seed storage protein genes. 10. Effect of light on plant development. 11. Flowering. 12. Breeding systems. 13. Reaction of plant to changes in the environment. 14. The molecular biology of disease and pest resistance . IV PART FOUR: An introduction to plant biotechnology. 15. Transgenic plants. 16. Prospects for plant biotechnology.