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Papers by alan berry

Biochemical Society Transactions, 1994

Molecular model of the predicted signal-binding site Models were constructed on New England Biogr... more Molecular model of the predicted signal-binding site Models were constructed on New England Biographics Promodeler I software, by residue replacement of the SRP sequences of the h2 and h3 helices on a classical a-helix, and by residue replacement of a p-strand found in the crystal structure of /l-lactoglobulin (residues 47-58) with residues I-I2 of the rat pre-elastase signal sequence. across the bilayer could then take place, possibly through a channel comprising, at least in part, the sec61 protein [ 121 and/or a 43 kDa integral membrane protein previously shown as a component of rough microsomes that cross-links to a photoreactive signal peptide [ 131. The signal sequence would finally be proteolytically removed by signal peptidase in the lumen of the ER.

Proceedings / ... International Conference on Intelligent Systems for Molecular Biology ; ISMB. International Conference on Intelligent Systems for Molecular Biology, 1994

An automated procedure for protein design by optimization of a sequence-structure quality has bee... more An automated procedure for protein design by optimization of a sequence-structure quality has been developed. The method selects a statistically optimal sequence for a particular structure, on the assumption that such a protein will adopt the desired structure. We present two optimization algorithms: one provides an exact optimization while the other uses a combinatorial technique for comparatively rapid results. Both are suitable for massively parallel computers. A prototype system was used to design sequences which should adopt the four-helix bundle conformation of myohemerythrin. These appear satisfactory to secondary structure and profile analysis. Detailed inspection reveals that the sequences are generally plausible but, as expected, lack some specific structural features. The design parameters provide some insight into the general determinants of protein structure.

Structure, 1996

Background: Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite c... more Background: Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. Results: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 Å resolution. The asymmetric unit is one subunit which presents a familiar fold, the (␣/␤) 8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 Å apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. Conclusions: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Protein Science, 2008

Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginin... more Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginine-specific a-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-"C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class I1 FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K,,, for fructose 1.6-bisphosphate. Comparatively small differences in the inhibitor constant K, for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (111) revealed that the K , for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

Protein Science, 1994

We have developed a general quantitative methodology for designing proteins de novo, which automa... more We have developed a general quantitative methodology for designing proteins de novo, which automatically produces sequences for any given plausible protein structure. The method incorporates statistical information, a theoretical description of protein structure, and motifs described in the literature. A model system embodying a portion of the quantitative methodology has been used to design many protein sequences for the phage 434 Cro and fibronectin type 111 domain folds, as well as several other structures. Residue sequences selected by this prototype share no significant identity with any natural protein. Nonetheless, 3-dimensional models of the designed sequences appear generally plausible. When examined using secondary structure prediction methods and profile analysis, the designed sequences generally score considerably better than the natural ones. The designed sequences are also in reasonable agreement with a sequence template. This quantitative methodology is likely to be capable of successfully designing new proteins and yielding fundamental insights about the determinants of protein structure.

Protein Science, 1994

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD ... more The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of l6.8%), of the wild-type enzyme ligated with NADP (2.OA, 20.8%), of the NAD-dependent mutant (1.74A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NADbinding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.

Protein Engineering Design and Selection, 2004

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following... more Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T 50) of the most stable variant, 4-43D6, is increased by 11-12 C over the wild-type enzymes and the half-life of activity at 53 C is increased $190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.

Proceedings of the National Academy of Sciences, 1991

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CA... more Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypa...

Molecular Microbiology, 2010

Biographical Memoirs of Fellows of the Royal Society, 2018

Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He... more Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He undertook his academic career at the University of Cambridge, holding positions as lecturer, reader, chair and head of the Department of Biochemistry, as well as becoming Master of St John's College. Perham published close to 300 scientific papers on protein structure and function, with a focus on mechanistic enzymology, particularly how large multienzyme complexes and flavin-containing enzymes work. He is most renowned for determining how reactive intermediates are transferred between enzyme active sites, for alterations of coenzyme and substrate specificity by his pioneering use of protein engineering and for developing protein display methodologies. Married to Nancy Lane-Perham, and with their two children, Perham enjoyed a full and active life in Cambridge and St John's College. He was a keen participator and supporter of sport and enjoyed art, literature, theatre and music....

Biochemical Society Transactions, 1994

Molecular model of the predicted signal-binding site Models were constructed on New England Biogr... more Molecular model of the predicted signal-binding site Models were constructed on New England Biographics Promodeler I software, by residue replacement of the SRP sequences of the h2 and h3 helices on a classical a-helix, and by residue replacement of a p-strand found in the crystal structure of /l-lactoglobulin (residues 47-58) with residues I-I2 of the rat pre-elastase signal sequence. across the bilayer could then take place, possibly through a channel comprising, at least in part, the sec61 protein [ 121 and/or a 43 kDa integral membrane protein previously shown as a component of rough microsomes that cross-links to a photoreactive signal peptide [ 131. The signal sequence would finally be proteolytically removed by signal peptidase in the lumen of the ER.

Proceedings / ... International Conference on Intelligent Systems for Molecular Biology ; ISMB. International Conference on Intelligent Systems for Molecular Biology, 1994

An automated procedure for protein design by optimization of a sequence-structure quality has bee... more An automated procedure for protein design by optimization of a sequence-structure quality has been developed. The method selects a statistically optimal sequence for a particular structure, on the assumption that such a protein will adopt the desired structure. We present two optimization algorithms: one provides an exact optimization while the other uses a combinatorial technique for comparatively rapid results. Both are suitable for massively parallel computers. A prototype system was used to design sequences which should adopt the four-helix bundle conformation of myohemerythrin. These appear satisfactory to secondary structure and profile analysis. Detailed inspection reveals that the sequences are generally plausible but, as expected, lack some specific structural features. The design parameters provide some insight into the general determinants of protein structure.

Structure, 1996

Background: Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite c... more Background: Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. Results: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 Å resolution. The asymmetric unit is one subunit which presents a familiar fold, the (␣/␤) 8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 Å apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. Conclusions: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Protein Science, 2008

Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginin... more Treatment of the Class 11 fructose-1.6-bisphosphate aldolase of Escherichia coli with the arginine-specific a-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme. The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate. Modification with [7-"C] phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme. Sequence alignment of the eight known Class I1 FBP-aldolases shows that only one arginine residue is conserved in all the known sequences. This residue, Arg-331, was mutated to either alanine or glutamic acid. The mutant enzymes were much less susceptible to inactivation by phenylglyoxal. Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K,,, for fructose 1.6-bisphosphate. Comparatively small differences in the inhibitor constant K, for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes. In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor. Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (111) revealed that the K , for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased. Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

Protein Science, 1994

We have developed a general quantitative methodology for designing proteins de novo, which automa... more We have developed a general quantitative methodology for designing proteins de novo, which automatically produces sequences for any given plausible protein structure. The method incorporates statistical information, a theoretical description of protein structure, and motifs described in the literature. A model system embodying a portion of the quantitative methodology has been used to design many protein sequences for the phage 434 Cro and fibronectin type 111 domain folds, as well as several other structures. Residue sequences selected by this prototype share no significant identity with any natural protein. Nonetheless, 3-dimensional models of the designed sequences appear generally plausible. When examined using secondary structure prediction methods and profile analysis, the designed sequences generally score considerably better than the natural ones. The designed sequences are also in reasonable agreement with a sequence template. This quantitative methodology is likely to be capable of successfully designing new proteins and yielding fundamental insights about the determinants of protein structure.

Protein Science, 1994

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD ... more The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of l6.8%), of the wild-type enzyme ligated with NADP (2.OA, 20.8%), of the NAD-dependent mutant (1.74A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NADbinding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.

Protein Engineering Design and Selection, 2004

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following... more Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T 50) of the most stable variant, 4-43D6, is increased by 11-12 C over the wild-type enzymes and the half-life of activity at 53 C is increased $190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.

Proceedings of the National Academy of Sciences, 1991

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CA... more Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypa...

Molecular Microbiology, 2010

Biographical Memoirs of Fellows of the Royal Society, 2018

Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He... more Richard Nelson Perham, FRS, FMedSci, FRSA, was a British professor of structural biochemistry. He undertook his academic career at the University of Cambridge, holding positions as lecturer, reader, chair and head of the Department of Biochemistry, as well as becoming Master of St John's College. Perham published close to 300 scientific papers on protein structure and function, with a focus on mechanistic enzymology, particularly how large multienzyme complexes and flavin-containing enzymes work. He is most renowned for determining how reactive intermediates are transferred between enzyme active sites, for alterations of coenzyme and substrate specificity by his pioneering use of protein engineering and for developing protein display methodologies. Married to Nancy Lane-Perham, and with their two children, Perham enjoyed a full and active life in Cambridge and St John's College. He was a keen participator and supporter of sport and enjoyed art, literature, theatre and music....