Site‐directed mutagenesis to facilitate X‐ray structural studies of <i>leuconostoc mesenteroides</i> glucose 6‐phosphate dehydrogenase

Adams, Margaret; Basak, A.K.; Gover, S.; Rowland, Paul; Levy, Haim

doi:10.1002/pro.5560020515

Cited by 9 publications

(13 citation statements)

References 26 publications

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“…It has been apparent for some time that numerous mutations underlying G6PD class I variants cluster in exon 10, and this is again true for both the class I variants in this series. Although it had been suggested that this region corresponds to the NADP-binding site, recent evidence from crystallographic analysis (Adams et al, 1993; Naylor et a/, unpublished observation) does not support this view. It is likely instead that this region corresponds to the subunit contact, and this makes it highly critical for the stability of the enzymatically active dimer.…”

Section: Discussionmentioning

confidence: 96%

Molecular genetics of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

et al. 1995

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In order to explore the nature of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain, we have analysed the G6PD gene in 11 unrelated Spanish G6PD-deficient males and their relatives by using the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with a direct PCR-sequencing procedure and PCR-restriction enzyme (RE) analysis. We have identified eight different missense mutations, six of which have been reported in previously described G6PD variants. In nine patients who had presented with acute favism we found the following mutations: G6PD A-376G-202A (four cases), G6PD Union1360T (two cases), G6PD Mediterranean563T (one case) and G6PD Aures143C (one case). In the remaining patient a novel A to G transition was found at nucleotide position 209 which has not been reported in any other ethnic group. This mutation results in a (70) Tyr to Cys substitution and the resulting G6PD variant was biochemically characterized and designated as G6PD Murcia. This new mutation creates a Bsp 1286I recognition site which enabled us to rapidly detect it by PCR-RE analysis. In two patients with chronic non-spherocytic haemolytic anaemia (CNSHA) we found the underlying genetic defects, as had been noted previously, to be located within a cluster of mutations in exon 10. One of them had the T to C transition at nucleotide 1153, causing a (385) Cys to Arg substitution, previously described in G6PD Tomah. The other, previously reported as having a variant called G6PD Clinic, has a G to A transition at nucleotide 1215 that produces a (405) Met to Ile substitution, thus confirming that G6PD Clinic is a new class I variant.

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Section: Discussionmentioning

confidence: 96%

Molecular genetics of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

et al. 1995

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“…The mutation Cys→Ala could be the perfect mutation of enhancing crystalizability according to our hypothesis in this study, which is reported as successful enhancement in the study [38]. The mutations of X→Cys are believed to enhance crystallizability for obtaining useful heavy-atom derivation [26], [43]–[45]. However, all mutations of X→Cys in these studies [26], [43]–[45] could not successfully improve crystallizability.…”

Section: Resultsmentioning

confidence: 61%

“…The mutations of X→Cys are believed to enhance crystallizability for obtaining useful heavy-atom derivation [26], [43]–[45]. However, all mutations of X→Cys in these studies [26], [43]–[45] could not successfully improve crystallizability. This scenario is reasonable according to our hypothesis.…”

Section: Resultsmentioning

confidence: 88%

“…Therefore, the mutations of Ser→X should increase the probability of enhancing protein crystallization for the same reason with Cys. We found the mutations of Ser→Cys for obtaining useful heavy-atom derivatives to enhance crystallizability [43]–[45]. However, all these mutations of Ser→Cys in the studies [43]–[45] fail to increase the crystallizability.…”

Section: Resultsmentioning

confidence: 89%

“…We found the mutations of Ser→Cys for obtaining useful heavy-atom derivatives to enhance crystallizability [43]–[45]. However, all these mutations of Ser→Cys in the studies [43]–[45] fail to increase the crystallizability. It might be reasonable that the mutation Cys→X demonstrated the high probability of enhancing protein crystallization [37]–[42].…”

Section: Resultsmentioning

confidence: 89%

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SCMCRYS: Predicting Protein Crystallization Using an Ensemble Scoring Card Method with Estimating Propensity Scores of P-Collocated Amino Acid Pairs

et al. 2013

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Existing methods for predicting protein crystallization obtain high accuracy using various types of complemented features and complex ensemble classifiers, such as support vector machine (SVM) and Random Forest classifiers. It is desirable to develop a simple and easily interpretable prediction method with informative sequence features to provide insights into protein crystallization. This study proposes an ensemble method, SCMCRYS, to predict protein crystallization, for which each classifier is built by using a scoring card method (SCM) with estimating propensity scores of p-collocated amino acid (AA) pairs (p = 0 for a dipeptide). The SCM classifier determines the crystallization of a sequence according to a weighted-sum score. The weights are the composition of the p-collocated AA pairs, and the propensity scores of these AA pairs are estimated using a statistic with optimization approach. SCMCRYS predicts the crystallization using a simple voting method from a number of SCM classifiers. The experimental results show that the single SCM classifier utilizing dipeptide composition with accuracy of 73.90% is comparable to the best previously-developed SVM-based classifier, SVM_POLY (74.6%), and our proposed SVM-based classifier utilizing the same dipeptide composition (77.55%). The SCMCRYS method with accuracy of 76.1% is comparable to the state-of-the-art ensemble methods PPCpred (76.8%) and RFCRYS (80.0%), which used the SVM and Random Forest classifiers, respectively. This study also investigates mutagenesis analysis based on SCM and the result reveals the hypothesis that the mutagenesis of surface residues Ala and Cys has large and small probabilities of enhancing protein crystallizability considering the estimated scores of crystallizability and solubility, melting point, molecular weight and conformational entropy of amino acids in a generalized condition. The propensity scores of amino acids and dipeptides for estimating the protein crystallizability can aid biologists in designing mutation of surface residues to enhance protein crystallizability. The source code of SCMCRYS is available at http://iclab.life.nctu.edu.tw/SCMCRYS/.

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A new lease of life for an old enzyme

Martini

Ursini

1996

BioEssays

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We review here some recent data about glucose-6-phosphate dehydrogenase (G6PD), the first and key regulatory enzyme of the pentose phosphate pathway. New evidence has been presented to suggest that malaria is a selective agent for G6PD deficiency, which is the most common enzymopathy in man, and that G6PD deficiency, generally considered to be a mild and benign condition, is significantly disadvantageous in certain environmental conditions. At the molecular level, the enzyme structure has recently been elucidated and mechanisms regulating G6PD gene expression have been determined. A G6PD knock-out mutation introduced in mouse cells makes them exquisitely sensitive to oxidative stress, indicating that this ubiquitous metabolic enzyme has a major role in the defence against oxidative stress, even in eukaryotic nucleated cells, which have several alternative routes for providing the same protection. Because of the high prevalence of G6PD deficiency in many populations, it is expected that these findings will prompt further studies to ascertain the putative role of G6PD deficiency in conditions such as carcinogenesis and ageing.

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Site‐directed mutagenesis to facilitate X‐ray structural studies of leuconostoc mesenteroides glucose 6‐phosphate dehydrogenase

Cited by 9 publications

References 26 publications

Molecular genetics of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

Molecular genetics of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Spain: identification of two new point mutations in the G6PD gene

SCMCRYS: Predicting Protein Crystallization Using an Ensemble Scoring Card Method with Estimating Propensity Scores of P-Collocated Amino Acid Pairs

A new lease of life for an old enzyme

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