Murtaza Alibhai - Academia.edu (original) (raw)

Papers by Murtaza Alibhai

J Allerg Clin Immunol, 2000

Journal of Allergy and Clinical Immunology, 2000

Journal of Allergy and Clinical Immunology, 2000

Biochemistry Usa, Feb 1, 1994

The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrat... more The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steadystate and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the K , values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the K , values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The ka,/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased k,,lK, value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NHd+) and the transition state for phosphoryl transfer while the E327A-Mn2+-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3. Glu-327 participates in lowering the free energy of the transition state involved in formation of the positively charged tetrahedral adduct resulting from the condensation of y-glutamyl phosphate and NH3.

Handbook of Plant Biotechnology, 2004

The role of herbicides in agriculture entered a new era with the introduction of glyphosate-resis... more The role of herbicides in agriculture entered a new era with the introduction of glyphosate-resistant soybeans in 1995 (Padgette et al., 1995). Engineered crop resistance has revolutionised agronomic practices by allowing in-crop use of broad-spectrum herbicides such as glyphosate and ...

Protein Science, 2008

In order to understand the nature of ATP and t-glutamate binding to glutamine synthetase, and the... more In order to understand the nature of ATP and t-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg --t Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the K,,, values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine) (DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in K,,, value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kc,, than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar K,,, values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA). The R339C mutant, upon modification with BPA, had a greater kc,, than WT enzyme; however, the R359C mutant did not show significant regeneration of activity with this reagent. The data are consistent with X-ray crystallographic studies showing Arg 339 and Arg 359 at the active site of glutamine synthetase (Liaw SH, Eisenberg D, 1994, Biochemistry 33:675-681) interacting with ATP, glutamate, and intermediates along the catalytic pathway. Because enzyme activity could be restored for the R339C mutant by monofunctional (amine) and bifunctional (amidine) reagents, Arg 339 most likely interacts with substrates in a monodentate fashion. Conversely, Arg 359 seems to interact bifunctionally with substrates because covalent modification of R359C with BPA did not lead to a significant regain of catalytic activity.

Proceedings of the National Academy of Sciences, 2005

Glyphosate is a broad-spectrum herbicide used for the control of weeds in glyphosate-resistant cr... more Glyphosate is a broad-spectrum herbicide used for the control of weeds in glyphosate-resistant crops. Glyphosate inhibits 5-enolpyruvyl shikimate 3-phosphate synthase, a key enzyme in the synthesis of aromatic amino acids in plants, fungi, and bacteria. Studies with glyphosate-resistant wheat have shown that glyphosate provided both preventive and curative activities against Puccinia striiformis f. sp. tritici and Puccinia triticina, which cause stripe and leaf rusts, respectively, in wheat. Growth-chamber studies demonstrated wheat rust control at multiple plant growth stages with a glyphosate spray dose typically recommended for weed control. Rust control was absent in formulation controls without glyphosate, dependent on systemic glyphosate concentrations in leaf tissues, and not mediated through induction of four common systemic acquired resistance genes. A field test with endemic stripe rust inoculum confirmed the activities of glyphosate pre-and postinfestation. Preliminary greenhouse studies also demonstrated that application of glyphosate in glyphosate-resistant soybeans suppressed Asian soybean rust, caused by Phakopsora pachyrhizi.

Crop Science, 2007

We report the generation of transgenic soybean [Glycine max (L.) Merr.] via Agrobacterium-mediate... more We report the generation of transgenic soybean [Glycine max (L.) Merr.] via Agrobacterium-mediated gene transfers of a cry1A gene (tic107) from Bacillus thuringiensis (Bt) that exhibits a high degree of resistance against the lepidopteran pests Pseudoplusia includens (Walker) (soybean looper), Helicoverpa zea (Boddie) (soybean podworm), and Anticarsia gemmatalis Hü bner (velvetbean caterpillar). Three transgenic soybean lines (862, 726, and 781) were evaluated for expression, molecular composition, efficacy against target pests, and agronomic characteristics. We have designed and tested an expression cassette that consistently and reproducibly generates transgenic soybeans that accumulate the tic107 protein at levels as high as 6.12 mg mg 21 of total extractable protein. Expression levels of this magnitude of a Bt insecticidal protein have never been reported in soybean. Previous reports have indicated expression levels 100-fold lower in the highest-expressing lines. In addition, the phenotypes of these high-expressing lines were indistinguishable from their negative segregant and the transformation parent (Asgrow var. 'A3237'). Insect bioassay data demonstrate complete protection against soybean looper, soybean podworm, and velvetbean caterpillar when negative controls exhibited defoliation as high as 98%. Unlike previous reports of transgenic soybeans, we report here highly efficacious, single-copy, and normal phenotypes of transgenic soybean plants containing the highly expressed cry1A gene.

Biochemistry, 1994

The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrat... more The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steadystate and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the K , values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the K , values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The ka,/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased k,,lK, value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NHd+) and the transition state for phosphoryl transfer while the E327A-Mn2+-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3. Glu-327 participates in lowering the free energy of the transition state involved in formation of the positively charged tetrahedral adduct resulting from the condensation of y-glutamyl phosphate and NH3.

Applied and Environmental Microbiology, 2011

Terminal olefins (1-alkenes) are natural products that have important industrial applications as ... more Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleT JE ), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleT JE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in ␣and/or ␤-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

Biochemistry, 2003

Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total so... more Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

J Allerg Clin Immunol, 2000

Journal of Allergy and Clinical Immunology, 2000

Journal of Allergy and Clinical Immunology, 2000

Biochemistry Usa, Feb 1, 1994

The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrat... more The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steadystate and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the K , values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the K , values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The ka,/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased k,,lK, value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NHd+) and the transition state for phosphoryl transfer while the E327A-Mn2+-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3. Glu-327 participates in lowering the free energy of the transition state involved in formation of the positively charged tetrahedral adduct resulting from the condensation of y-glutamyl phosphate and NH3.

Handbook of Plant Biotechnology, 2004

The role of herbicides in agriculture entered a new era with the introduction of glyphosate-resis... more The role of herbicides in agriculture entered a new era with the introduction of glyphosate-resistant soybeans in 1995 (Padgette et al., 1995). Engineered crop resistance has revolutionised agronomic practices by allowing in-crop use of broad-spectrum herbicides such as glyphosate and ...

Protein Science, 2008

In order to understand the nature of ATP and t-glutamate binding to glutamine synthetase, and the... more In order to understand the nature of ATP and t-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg --t Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the K,,, values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine) (DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in K,,, value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kc,, than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar K,,, values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA). The R339C mutant, upon modification with BPA, had a greater kc,, than WT enzyme; however, the R359C mutant did not show significant regeneration of activity with this reagent. The data are consistent with X-ray crystallographic studies showing Arg 339 and Arg 359 at the active site of glutamine synthetase (Liaw SH, Eisenberg D, 1994, Biochemistry 33:675-681) interacting with ATP, glutamate, and intermediates along the catalytic pathway. Because enzyme activity could be restored for the R339C mutant by monofunctional (amine) and bifunctional (amidine) reagents, Arg 339 most likely interacts with substrates in a monodentate fashion. Conversely, Arg 359 seems to interact bifunctionally with substrates because covalent modification of R359C with BPA did not lead to a significant regain of catalytic activity.

Proceedings of the National Academy of Sciences, 2005

Glyphosate is a broad-spectrum herbicide used for the control of weeds in glyphosate-resistant cr... more Glyphosate is a broad-spectrum herbicide used for the control of weeds in glyphosate-resistant crops. Glyphosate inhibits 5-enolpyruvyl shikimate 3-phosphate synthase, a key enzyme in the synthesis of aromatic amino acids in plants, fungi, and bacteria. Studies with glyphosate-resistant wheat have shown that glyphosate provided both preventive and curative activities against Puccinia striiformis f. sp. tritici and Puccinia triticina, which cause stripe and leaf rusts, respectively, in wheat. Growth-chamber studies demonstrated wheat rust control at multiple plant growth stages with a glyphosate spray dose typically recommended for weed control. Rust control was absent in formulation controls without glyphosate, dependent on systemic glyphosate concentrations in leaf tissues, and not mediated through induction of four common systemic acquired resistance genes. A field test with endemic stripe rust inoculum confirmed the activities of glyphosate pre-and postinfestation. Preliminary greenhouse studies also demonstrated that application of glyphosate in glyphosate-resistant soybeans suppressed Asian soybean rust, caused by Phakopsora pachyrhizi.

Crop Science, 2007

We report the generation of transgenic soybean [Glycine max (L.) Merr.] via Agrobacterium-mediate... more We report the generation of transgenic soybean [Glycine max (L.) Merr.] via Agrobacterium-mediated gene transfers of a cry1A gene (tic107) from Bacillus thuringiensis (Bt) that exhibits a high degree of resistance against the lepidopteran pests Pseudoplusia includens (Walker) (soybean looper), Helicoverpa zea (Boddie) (soybean podworm), and Anticarsia gemmatalis Hü bner (velvetbean caterpillar). Three transgenic soybean lines (862, 726, and 781) were evaluated for expression, molecular composition, efficacy against target pests, and agronomic characteristics. We have designed and tested an expression cassette that consistently and reproducibly generates transgenic soybeans that accumulate the tic107 protein at levels as high as 6.12 mg mg 21 of total extractable protein. Expression levels of this magnitude of a Bt insecticidal protein have never been reported in soybean. Previous reports have indicated expression levels 100-fold lower in the highest-expressing lines. In addition, the phenotypes of these high-expressing lines were indistinguishable from their negative segregant and the transformation parent (Asgrow var. 'A3237'). Insect bioassay data demonstrate complete protection against soybean looper, soybean podworm, and velvetbean caterpillar when negative controls exhibited defoliation as high as 98%. Unlike previous reports of transgenic soybeans, we report here highly efficacious, single-copy, and normal phenotypes of transgenic soybean plants containing the highly expressed cry1A gene.

Biochemistry, 1994

The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrat... more The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steadystate and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the K , values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the K , values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The ka,/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased k,,lK, value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NHd+) and the transition state for phosphoryl transfer while the E327A-Mn2+-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3. Glu-327 participates in lowering the free energy of the transition state involved in formation of the positively charged tetrahedral adduct resulting from the condensation of y-glutamyl phosphate and NH3.

Applied and Environmental Microbiology, 2011

Terminal olefins (1-alkenes) are natural products that have important industrial applications as ... more Terminal olefins (1-alkenes) are natural products that have important industrial applications as both fuels and chemicals. However, their biosynthesis has been largely unexplored. We describe a group of bacteria, Jeotgalicoccus spp., which synthesize terminal olefins, in particular 18-methyl-1-nonadecene and 17-methyl-1nonadecene. These olefins are derived from intermediates of fatty acid biosynthesis, and the key enzyme in Jeotgalicoccus sp. ATCC 8456 is a terminal olefin-forming fatty acid decarboxylase. This enzyme, Jeotgalicoccus sp. OleT (OleT JE ), was identified by purification from cell lysates, and its encoding gene was identified from a draft genome sequence of Jeotgalicoccus sp. ATCC 8456 using reverse genetics. Heterologous expression of the identified gene conferred olefin biosynthesis to Escherichia coli. OleT JE is a P450 from the cyp152 family, which includes bacterial fatty acid hydroxylases. Some cyp152 P450 enzymes have the ability to decarboxylate and to hydroxylate fatty acids (in ␣and/or ␤-position), suggesting a common reaction intermediate in their catalytic mechanism and specific structural determinants that favor one reaction over the other. The discovery of these terminal olefin-forming P450 enzymes represents a third biosynthetic pathway (in addition to alkane and long-chain olefin biosynthesis) to convert fatty acid intermediates into hydrocarbons. Olefin-forming fatty acid decarboxylation is a novel reaction that can now be added to the catalytic repertoire of the versatile cytochrome P450 enzyme family.

Biochemistry, 2003

Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total so... more Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.