A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Evans, G.L.; Schuldt, Linda; Griffin, Michael D. W.; Devenish, Sean R. A.; Pearce, F. Grant; Perugini, Matthew A.; Dobson, Renwick C. J.; Jameson, Geoffrey B.; Weiss, M.S.; Gerrard, Juliet A.

doi:10.1016/j.abb.2011.05.014

Cited by 16 publications

(9 citation statements)

References 39 publications

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“…The presence of pyruvate in the growth media and purification buffers was investigated. While unsuccessful for the PH3 variants, this method has proven successful in stabilizing oligomeric state disrupting variants and increasing overall yields of the related DHDPS family of enzymes [13-15]. Further attempts to stabilize the protein by the addition of glycerol and/or DTT to the buffer also proved unsuccessful.…”

Section: Resultsmentioning

confidence: 99%

4-Hydroxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and glyoxylate reductase inhibition

Riedel

Knight

Murray

et al. 2012

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Mutations in the gene encoding for 4-hydroxy-2-oxoglutarate aldolase (HOGA) are associated with an excessive production of oxalate in Primary Hyperoxaluria type 3 (PH3). This enzyme is the final step of the hydroxyproline degradation pathway within the mitochondria and catalyzes the cleavage of 4-hydroxy-2-oxoglutarate (HOG) to pyruvate and glyoxylate. No analyses have been performed to assess the consequences of the mutations identified, particularly for those variants that produce either full-length or nearly full-length proteins. In this study, the expression, stability, and activity of nine PH3 human HOGA variants were examined. Using recombinant protein produced in Escherichia coli as well as transfected Chinese hamster ovary (CHO) cells, it was found that all nine PH3 variants are quite unstable, have a tendency to aggregate, and retain no measurable activity. A buildup of HOG was confirmed in the urine, sera and liver samples from PH3 patients. To determine how HOG is cleaved in the absence of HOGA activity, the ability of N-acetylneuraminate aldolase (NAL) to cleave HOG was evaluated. NAL showed minimal activity towards HOG. Whether the expected buildup of HOG in mitochondria could inhibit glyoxylate reductase (GR), the enzyme mutated in PH2, was also evaluated. GR was inhibited by HOG but not by 2-hydroxyglutarate or 2-oxoglutarate. Thus, one hypothetical component of the molecular basis for the excessive oxalate production in PH3 appears to be the inhibition of GR by HOG, resulting in a phenotype similar to PH2.

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Section: Resultsmentioning

confidence: 99%

4-Hydroxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and glyoxylate reductase inhibition

Riedel

Knight

Murray

et al. 2012

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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“…The importance of protein oligomerization can be studied by comparing the catalytic properties of the monomeric and oligomeric enzyme forms. An approach to examine the role of quaternary structures is to artificially create the monomers by identifying putative hot spot residues in the oligomerization interface and harnessing these hot spot residues to design mutants with perturbed intersubunit interactions . In the present study, we followed a complementary approach by identifying hot spot residues in the dimer interface of known AnPRT variants by means of a comprehensive sequence comparison.…”

Section: Discussionmentioning

confidence: 70%

“…An approach to examine the role of quaternary structures is to artificially create the monomers by identifying putative hot spot residues in the oligomerization interface and harnessing these hot spot residues to design mutants with perturbed intersubunit interactions. 10,[53][54][55][56] In the present study, we followed a complementary approach by identifying hot spot residues in the dimer interface of known AnPRT variants by means of a comprehensive sequence comparison. According to the computer-based analysis (PISA service), three hydrophobic residues, I36 (numbering according to ssAnPRT) and M47 in helix α3, and I167 in helix α8, are Abbreviations: EV, elution volume obtained in analytical gel-filtration chromatography (GFC); GFC MW app , apparent molecular mass as deduced from analytical GFC; TDA MW app , weight average of samples as deduced from TDA analysis; MW calc , molecular mass calculated from the amino acid sequence for monomer or dimer; ne, not evaluable.…”

Section: Discussionmentioning

confidence: 99%

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

et al. 2019

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It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic “hot spot” amino acids in the protein‐protein interface that were predicted to be essential for self‐association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer‐dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

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“…Two such dimers then unite to create a tetramer via interactions at the 'weak-dimer' interface where weaker bonding interactions exist (Perugini et al, 2005;Griffin et al, 2008Griffin et al, , 2010. Recently, homodimers and dimeric mutants of DHDPS have also surfaced that exhibit high levels of activity (Burgess et al, 2008;Girish et al, 2008;Wubben et al, 2010;Evans et al, 2011;Kaur et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

“…The structure of DHDPS has been investigated in several bacteria including Agrobacterium tumefaciens , Bacillus anthracis (Blagova et al, 2006;Voss et al, 2009Voss et al, , 2010, Clostridium botulinum , Corynebacterium glutamicum (Rice et al, 2008), Escherichia coli (Mirwaldt et al, 1995;Dobson, Griffin et al, 2005), Hahella chejuensis (Kang et al, 2010), Methanococcus jannaschii (Padmanabhan et al, 2009), Mycobacterium tuberculosis (Kefala et al, 2008;Evans et al, 2011), Neisseria meningitidis (Devenish et al, 2009), Pseudomonas aeruginosa (Kaur et al, 2011), Shewenella benthica (Wubben et al, 2010), Staphylococcus aureus (Burgess et al, 2008;Girish et al, 2008), Streptococcus pneumoniae (Sibarani et al, 2010) and Thermotoga maritima (Pearce et al, 2006). The enzyme typically forms a homotetramer that is described as a dimer of dimers.…”

Section: Introductionmentioning

confidence: 99%

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

et al. 2013

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Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of highmolecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 Å resolution. The crystal lattice belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 89.31, c = 290.18 Å , and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.

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A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

Cited by 16 publications

References 39 publications

4-Hydroxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and glyoxylate reductase inhibition

4-Hydroxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and glyoxylate reductase inhibition

Prediction of quaternary structure by analysis of hot spot residues in protein‐protein interfaces: the case of anthranilate phosphoribosyltransferases

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

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