Construction of a high-copy “ATG vector” for expression in Escherichia coli

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics is critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity/coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C distinguishable by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9Å. Notably, the coupling energy (ΔG I ) is anti-correlated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for about 70% of the energy difference for the ratio of k cat /K m for the two substrates between aldolase A and aldolase B. These non-additive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.Recent evidence shows dynamic fluctuations of structure are essential components of enzyme catalysis (1). While it is likely that most enzymes exhibit flexibility as part of the catalytic process, the role of this movement is enzyme specific. For example, in the case of dihydrofolate reductase (DHFR), backbone and side-chain motions are essential for cofactor binding, † This work was supported by Grants GM60616 (to D.R.T. and K.N.A.), DK065089 (to D.R.T), and Training Grant HL07291 (to J.A.P.) from the National Institutes of Health.*To whom correspondence may be addressed: Dean R. Tolan, Biology Department, 5 Cummington St., Boston MA 02215; (617) 353-5310 (tel), (617) 358-0338 (fax), email: tolan@bu.edu., Karen N. Allen, Department of Physiology and Biophysics, 715 Albany St., Boston University School of Medicine, Boston MA 02118; (617) 638-4398 (tel), (617) 638-4273 (fax), allen@med-xtal.bu.edu. ⊥ Present address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 185 Meeting Street, Box G-L2, Providence RI 02912 1 ISR, isozyme-specific residue; Fru 1,6-P 2 , fructose 1,6-bisphosphate; Fru 1-P, fructose 1-phosphate; CTR, C-terminal region; TSP, terminal surface patch; DSP, distal surface patch; DTNB, 5,5′dithiobis(2-nitrobenzoic acid). Recently, the importance of conformational flexibility has ...

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“…Full-length cassettes were subcloned into EcoR I and Hind III sites in pPB1 (30). The different expression constructs are described in Table 1.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Thermodynamic Analysis Shows Conformational Coupling and Dynamics Confer Substrate Specificity in Fructose-1,6-bisphosphate Aldolase

et al. 2007

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“…Creation of Stable Cell Lines-Plasmids used were pMSCV (Clontech), pcDNA 3.1-Myc (Invitrogen), pPB14 (rabbit aldolase A expression plasmid) (16), and pCL10-A1 (Clontech). The pMSCV-MycAldolase plasmid was constructed by PCR amplification of the rabbit aldolase A open reading frame from pPB14 (17) with primers containing restriction enzyme sites compatible for cloning into pcDNA3.1-Myc.…”

Section: Methodsmentioning

confidence: 99%

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

Lew

Tolan

2012

Journal of Biological Chemistry

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Background: Due to renewed interest in the Warburg effect, glycolytic enzymes have garnered interest as therapeutic targets for cancer. Results: Proliferation of transformed cell lines is halted upon aldolase knockdown using RNAi, an effect not seen upon knockdown of other glycolytic enzymes. Conclusion: Aldolase knockdown inhibits proliferation through a non-glycolytic function, likely affecting cytokinesis. Significance: Non-glycolytic aldolase functions represent a new potential target for cancer therapeutics.

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“…Underlined nucleotides refer to the codon that has been changed from that of the wild-type aldolase A. The mutant enzyme AB_All was generated via multiple site-directed mutagenesis using overlapping oligodeoxyribonucleotides (32) that complement the high copy ATG vector expressing rabbit aldolase A, pPB14 (33). This construction involved three steps.…”

Section: Methodsmentioning

confidence: 99%

Spatial Clustering of Isozyme-specific Residues Reveals Unlikely Determinants of Isozyme Specificity in Fructose-1,6-bisphosphate Aldolase

Pezza

Choi

Berardini

et al. 2003

Journal of Biological Chemistry

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Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.Vertebrate fructose-1,6-bisphosphate aldolase is a ubiquitous tetrameric enzyme that catalyzes reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways (1). The enzyme catalyzes the reversible aldol cleavage of Fru 1,6-P 2 1 into two trioses, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. It also cleaves the structurally related sugar Fru 1-P into glyceraldehyde and dihydroxyacetone phosphate (2, 3).Vertebrate aldolases exist as three or more isozymes with different tissue distributions: aldolase A (expressed predominantly in muscle), aldolase B (expressed predominantly in liver), and aldolase C (expressed predominantly in brain) (1). The sequences of the mammalian isozymes are highly conserved, exhibiting 81% sequence identity between aldolases A and C (4). Aldolase B is slightly more divergent with ϳ70% sequence identity to both aldolases A and C (5). Consistent with this sequence similarity, isozymes A and C exhibit comparable kinetic properties. Aldolases A and C have evolved to perform the glycolytic reaction, Fru 1,6-P 2 cleavage, more efficiently than aldolase B as demonstrated by a 20 -30-fold higher k ...

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Construction of a high-copy “ATG vector” for expression in Escherichia coli

Cited by 24 publications

References 16 publications

Thermodynamic Analysis Shows Conformational Coupling and Dynamics Confer Substrate Specificity in Fructose-1,6-bisphosphate Aldolase

Thermodynamic Analysis Shows Conformational Coupling and Dynamics Confer Substrate Specificity in Fructose-1,6-bisphosphate Aldolase

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

Spatial Clustering of Isozyme-specific Residues Reveals Unlikely Determinants of Isozyme Specificity in Fructose-1,6-bisphosphate Aldolase

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