Use of site-directed mutagenesis to probe the role of Cys<sup>149</sup> in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Mougin, Annie; Corbier, Catherine; Soukri, Abdelaziz; Wonacott, A.; Branlant, Christiane; Branlant, Guy

doi:10.1093/protein/2.1.45

Cited by 41 publications

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“…strain PCC 6803 has been analyzed and found to have the structural features typical of other gap genes, including (i) the well-conserved sequence of the active site 152 ASCTTNCL 159 (according to the numbering of the predicted Synechocystis sp. strain PCC 6803 GAPDH2) around the C 154 (in boldface) that forms the thioester bond with the substrate in the catalysis (30) and (ii) the so-called S-loop region (residues 183 to 205) which is involved in the interaction between subunits and the binding of the nucleotide cofactor and displayed prokaryotic signatures (16). A proline residue of this region present in glycolytic GAPDHs, P 188 according to the numbering of Bacillus stearothermophilus GAPDH (5), has been postulated to be involved in pyridine nucleotide cofactor specificity conferring absolute specificity for NAD (12).…”

Section: Resultsmentioning

confidence: 99%

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

1997

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The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD-to NADPdependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.Phosphorylating D-glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) are enzymes involved in the central pathways of carbon metabolism that have been found in all organisms studied so far (16). Due to their key metabolic roles, these proteins and their corresponding gap genes have been well characterized in many organisms (8,16). In the case of the widespread glycolytic GAPDH, by far the best-studied member of this enzyme family, the knowledge of its structure has allowed directed mutagenesis of essential amino acid residues of the active site and the eventual detailed description of the enzymatic mechanism (12,18,44).Three distinct GAPDH enzymes with different subcellular localizations and performing diverse roles are present in photosynthetic eukaryotic organisms: (i) a typical NAD-dependent...

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Section: Resultsmentioning

confidence: 99%

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

1997

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“…This is a situation similar to that observed with phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. In this latter enzyme, it was shown that the transition was due to a charge transfer between the essential Cys-149 and the nicotinamide ring of the cofactor acting as an electron donor and electron acceptor, respectively (20,21). Therefore, it was reasonable to hypothesize that Ab 333 was due to a charge transfer between Cys-302 and the nicotinamide of NADP.…”

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Characterization of the Amino Acids Involved in Substrate Specificity of Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus mutans

Marchal¹,

Branlant²

2002

Journal of Biological Chemistry

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In order to address the molecular basis of the specificity of aldehyde dehydrogenase for aldehyde substrates, enzymatic characterization of the glyceraldehyde 3-phosphate (G3P) binding site of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans has been undertaken. In this work, residues Arg-124, Tyr-170, Arg-301, and Arg-459 were changed by site-directed mutagenesis and the catalytic properties of GAPN mutants investigated. Changing Tyr-170 into phenylalanine induces no major effect on k cat and K m for D-G3P in both acylation and deacylation steps. Substitutions of Arg-124 and Arg-301 by leucine and Arg-459 by isoleucine led to distinct effects on K m , on k cat , or on both. The ratelimiting step of the R124L GAPN remains deacylation. Pre-steady-state analysis and substrate isotope measurements show that hydride transfer remains rate-determining in acylation. Only the apparent affinity for D-G3P is decreased in both acylation and deacylation steps. Substitution of Arg-459 by isoleucine leads to a drastic effect on the catalytic efficiency by a factor of 10 5 . With this R459L GAPN, the rate-limiting step is prior to hydride transfer, and the K m of D-G3P is increased by at least 2 orders of magnitude. Binding of NADP leads to a time-dependent formation of a charge transfer transition at 333 nm between the pyridinium ring of NADP and the thiolate of Cys-302, which is not observed with the holo-wild type. Accessibility of Cys-302 is shown to be strongly decreased within the holostructure. The substitution of Arg-301 by leucine leads to an even more drastic effect with a change of the rate-limiting step similar to that observed for R459I GAPN. Taking into account the three-dimensional structure of GAPN from S. mutans and the data of the present study, it is proposed that 1) Tyr-170 is not essential for the catalytic event, 2) Arg-124 is only involved in stabilizing D-G3P binding via an interaction with the C-3 phosphate, and 3) Arg-301 and Arg-459 participate not only in D-G3P binding via interaction with C-3 phosphate but also in positioning efficiently D-G3P relative to Cys-302 within the ternary complex GAPN⅐NADP⅐D-G3P.Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) 1 catalyzes the irreversible oxidation of D-glyceraldehyde 3-phosphate (D-G3P) into 3-phosphoglycerate in the presence of NADP via a two-step chemical mechanism. It belongs to the aldehyde dehydrogenase (ALDH) superfamily, which oxidizes a wide variety of aldehydes into nonactivated or SCoA-activated acidic compounds (1-3). These enzymes play parts at several levels of cellular metabolism including anabolic and catabolic pathways, detoxification processes, and embryogenesis development. Their numerous functions probably explain why most ALDHs show a wide substrate specificity, except for the CoA-dependent ALDH. In mammals, ALDH class 1 shows preference for retinal but it can also oxidize aromatic and "long-chain" aldehydes with dissimilar catalytic efficiencies, whereas ALDHs that belong ...

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“…These strains were transformed with the plasmid pBS::EcogapA. This plasmid was constructed by cutting out the HindIII-BamHI 2,149-nucleotide fragment from phage M13mp9:EcogapA (38) and inserting it into plasmid BSIISK+ (Stratagene). The plasmid pBS::EcogapA contains the entire gapA gene with a 346-nucleotide upstream flanking region.…”

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The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32

Charpentier

Branlant

1994

J Bacteriol

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Escherichia coli D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is produced by the gapA gene and is structurally related to eukaryotic GAPDHs. These facts led to the proposal that the gapA gene originated by a horizontal transfer of genetic information. The yields and start sites of gapA mRNAs produced in various fermentation conditions and genetic contexts were analyzed by primer extension. The transcriptional regulatory region of the gapA gene was found to contain four promoter sequences, three recognized by the vegetative RNA polymerase Eff70 and one recognized by the heat shock RNA polymerase E&32. Transcription ofgapA by Eu32 is activated in the logarithmic phase under conditions of starvation and of heat shock Using a GAPDH-strain, we found that GAPDH production has a positive effect on cell growth at 43°C. Thus, E. coli GAPDH displays some features of heat shock proteins. One of the gapA promoter sequences transcribed by Eu70 is subject to catabolic repression. Another one has growth phase-dependent efficiency. This complex area of differentially regulated promoters allows the production of large amounts of gapA transcripts in a wide variety of environmental conditions. On the basis of these data, the present view of Eu32 RNA polymerase function has to be enlarged, and the various hypotheses on E. coli gapA gene origin have to be reexamined.The NAD+-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which converts D-glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate (22), is a key enzyme of the glycolytic and gluconeogenesis pathways. In eubacteria (5,14,17,46) and archaebacteria (4), the GAPDH gene (gap) belongs to a cluster of genes coding for several glycolytic enzymes: 3-phosphoglycerate kinase (pgk), class II fructose 1,6-bisphosphate aldolase (fda), and triosephosphate isomerase (tpi). A peculiar situation is observed in Escherichia coli, in which the gapB gene, located in this cluster, does not produce GAPDH activity (2, 25). The active gapA gene and the pgk gene are 22.6 min apart on the E. coli chromosome (7,50). In addition, the gapA gene codes for a protein which has stronger homology with mammalian GAPDHs than with bacterial enzymes (6). A eukaryotic origin has thus been proposed for the gapA gene: enterobacteria would have acquired the gapA gene through a horizontal transmission of genetic information, concomittant with the loss of activity of the gapB gene (2,16,36 recognized by the RNA polymerase holoenzyme EC70, which is formed by association of the holoenzyme with the major sigma factor, u70 (24). A few subsets of genes that respond to various environmental or nutritional changes require specific recognition by RNA polymerase associated with the alternative sigma factor u32 (21), crE (18,52), 0k54 (26), or as (39). The heat shock response mediated by the Eu32 RNA polymerase holoenzyme has been the subject of several studies (for reviews, see references 8 and 40). When E. coli cells growing at 30°C are shifted to 42°C, the rate of synthesis of the so-call...

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Use of site-directed mutagenesis to probe the role of Cys¹⁴⁹ in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Cited by 41 publications

References 0 publications

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Characterization of the Amino Acids Involved in Substrate Specificity of Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus mutans

The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32

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Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Cited by 41 publications

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Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803

Characterization of the Amino Acids Involved in Substrate Specificity of Nonphosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase from Streptococcus mutans

The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme E sigma 70 and by the heat shock RNA polymerase E sigma 32

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Use of site-directed mutagenesis to probe the role of Cys¹⁴⁹ in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase