Serine Hydroxymethyltransferase: Role of the Active Site Lysine in the Mechanism of the Enzyme

Schirch, Douglas; S, Delle Fratte; Iurescia, Sandra; Angelaccio, Sebastiana; Contestabile, Roberto; Bossa, Francesco; Schirch,

doi:10.1007/978-1-4615-2960-6_148

Cited by 12 publications

(21 citation statements)

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“…Mutation of K229 involved in forming a Schiff's base with PLP in Escherichia coli SHMT (eSHMT) [10] and of K256 in sheep liver cytosolic recombinant SHMT (rSHMT) [11] showed that this residue was essential for PLP binding and was not involved in proton abstraction [10,11], unlike in other PLP enzymes [12][13][14][15]. In addition, mutation of K256 leads to the dissociation of the tetramer to dimers in the case of rSHMT [11].…”

Section: Introductionmentioning

confidence: 99%

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5h-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 µM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5 % active. The

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Section: Introductionmentioning

confidence: 99%

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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“…They have been shown to increase the reactivity of the PLP 4Ј-aldehyde group, facilitating the formation of an external aldimine between the substrate and the PLP cofactor (3-9). In addition, lysine residues have also been shown to be involved in catalysis (4 -7, 10, 11), the formation of enzyme-substrate intermediates and product release (5,8,9), and cofactor binding (4,5,12).Although progress in the spectroscopic and kinetic features of T. denticola cystalysin has been made recently, the catalytic mechanism and the individual residues essential for enzyme activity remain to be elucidated. To understand the functional contribution of the active site lysine residue (Lys-238) to stages of the reaction catalyzed by cystalysin, we have changed Lys-238 of cystalysin to Ala or Arg and have studied the kinetic and spectroscopic properties of the mutants.…”

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confidence: 99%

Lysine 238 Is an Essential Residue for α,β-Elimination Catalyzed by Treponema denticola Cystalysin

Bertoldi

Cellini

D’Aguanno³

et al. 2003

Journal of Biological Chemistry

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Treponema denticola cystalysin is a pyridoxal 5-phosphate (PLP) enzyme that catalyzes the ␣,␤-elimination of L-cysteine to pyruvate, ammonia, and H 2 S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently ϳ50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 10 3 -and 7.5 ؋ 10 4 -fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the ␣,␤-elimination of L-cysteine and ␤-chloro-L-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the C␣-proton from the substrate and possibly as a general acid protonating the ␤-leaving group. Cystalysin utilizes pyridoxal 5Ј-phosphate (PLP)1 as its coenzyme and is categorized as a member of the ␣ subfamily of PLP-dependent enzymes that include aspartate aminotransferase. The cDNA from Treponema denticola has been heterologously cloned in Escherichia coli, the recombinant enzyme has been crystallized, and the three-dimensional structure solved (1). The PLP cofactor in the active site forms a Schiff base with the ⑀-amino group of Lys-238 of cystalysin (2). The spectrum of cystalysin exhibits absorption maxima at 418 and 320 nm, in addition to the protein band at 281 nm. The 418-nm band is due to the protonated internal aldimine, whereas the 320-nm band is due to a substituted aldamine. The apparent pK spec of this spectral transition is ϳ8.4 (2).Cystalysin catalyzes the ␣,␤-elimination of L-cysteine to generate pyruvate, ammonia, and H 2 S (3). A catalytic mechanism has been suggested in which, after transaldimination, the released Lys-238 abstracts the C␣ proton from the substrate with its deprotonated ⑀-amino group, producing a carbanionic intermediate stabilized as the characteristic quinonoid intermediate. Then, after reaching an optimal position within hydrogen bonding distance to S␥, Lys-238 protonates the sulfur atom, and the resulting thiol is released. Finally, reverse transaldimination of the PLP aminoacrylate takes place; Lys-238 attacks the C4Ј atom, converting the aminoacrylate into an iminoproprionate, which is released and hydrolyzed to pyruvate and ammonia (1). Recently, substrate specificity studies reveal that cystalysin displays ...

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“…However, little is known about specific promoter structure for H. methylovorum GM2, and further study will be necessary to define the promoter regions. mined with rabbit liver SHMT [25,261. As shown in Fig.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2

Miyata

Yoshida

Yamagata

et al. 1993

European Journal of Biochemistry

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The gene encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the one-carbon-compound assimilation of a methylotroph, Hyphomicrobium methylovorum GM2, and its flanking regions were isolated using a DNA fragment encoding Escherichia coli SHMT as a probe. Nucleotide sequencing of the recombinant plasmids revealed the SHMT gene codes for the 434-amino-acid protein with a calculated molecular mass of 46 068 Da. The amino-acid sequence of the enzyme showed identity to the sequences of the enzymes from E. coli (55%) and rabbit liver (44%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced to an SHMT-deficient E. coli mutant ME5427 (glyA-). The transformed E. coli cells expressed SHMT, which was immunologically and enzymologically indistinguishable from the enzyme isolated from H. methylovorum GM2.Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine, with tetrahydrofolate as the one-carbon ( C , ) acceptor. This enzyme activity is widely distributed in nature, being found in prokaryote and eukaryote. Its major functions are to provide glycine for protein and purine synthesis, and 5,lO-methylenetetrahydrofolate for the C , pool. Recently, primary structures of the enzymes from rabbit liver, Salmonella typhimurium, Bacillus stearothermophilus, Escherichia coli, Bradyrhizobium japonicum and Campilobacter jejuni have been determined [l -71. A number of methylotrophic bacteria possess the serine pathway for the assimilation of C , compounds, which provides precursors for the biosyntheses of cell components from C , substrates. In such bacteria, SHMT plays an important role as the first enzyme in the assimilation of C , compounds through the transfer of formaldehyde (via 5,lO-Correspondence to

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Serine Hydroxymethyltransferase: Role of the Active Site Lysine in the Mechanism of the Enzyme

Cited by 12 publications

References 7 publications

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Lysine 238 Is an Essential Residue for α,β-Elimination Catalyzed by Treponema denticola Cystalysin

Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2

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