Serine hydroxymethyltransferase from Escherichia coli: purification and properties

Schirch, Verne; Hopkins, Sam; Villar, Enrique; Angelaccio, Sebastiana

doi:10.1128/jb.163.1.1-7.1985

Cited by 165 publications

(72 citation statements)

References 20 publications

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“…where V max,MTHFD1 = 594,000 μM h −1 , K m,CHF = 10 μM 73 (CHF = 5,10-methenylTHF); parameters of the second equation are: V max,SHMT = 5200 μM h −1 , K m,THF = 50 μM and K m,Serine = 600 μM. 66,[73][74][75][76][77][78][79][80] Also described is the nonenzymatic generation of 5,10-methyleneTHF by the condensation of formaldehyde (HCHO) and THF (K 1 = 0.03 μM −1 h −1 ), 75,81 adopting a bimolecular kinetics (i.e., two reactants yielding to a singular product).…”

Section: Chfmentioning

confidence: 99%

Modeling cellular compartmentation in one‐carbon metabolism

Scotti

Stella

Shearer

et al. 2013

WIREs Mechanisms of Disease

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Folate-mediated one-carbon metabolism (FOCM) is associated with risk for numerous pathological states including birth defects, cancers, and chronic diseases. Although the enzymes that constitute the biological pathways have been well described and their interdependency through the shared use of folate cofactors appreciated, the biological mechanisms underlying disease etiologies remain elusive. The FOCM network is highly sensitive to nutritional status of several B-vitamins and numerous penetrant gene variants that alter network outputs, but current computational approaches do not fully capture the dynamics and stochastic noise of the system. Combining the stochastic approach with a rule-based representation will help model the intrinsic noise displayed by FOCM, address the limited flexibility of standard simulation methods for coarse-graining the FOCM-associated biochemical processes, and manage the combinatorial complexity emerging from reactions within FOCM that would otherwise be intractable.

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Section: Chfmentioning

confidence: 99%

Modeling cellular compartmentation in one‐carbon metabolism

Scotti

Stella

Shearer

et al. 2013

WIREs Mechanisms of Disease

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“…The rabbit and E. coli SHMTs were reported to bind to glycine and many l-and d-amino acids (e.g. l-serine, d-alanine and l-alanine), which Recombinant form of cytosolic enzyme [14] D,L-allo-threonine 1.5 1.34 Bacillus stearothermophilus [24] L-allo-threonine 0.4 ± 0.009 0.9 ± 0.002 Escherichia coli [11] D,L-allo-threonine 1.5 0.5 resulted in the formation of E-Q [5,35]. However, incubation of d-serine with these enzymes did not result in the formation of E-Q [5,35].…”

Section: Reaction Of Pvshmt With D-serinementioning

confidence: 99%

“…Besides catalyzing its main physiological reaction of SHMT activity, the enzyme is known to catalyze a broad range of reactions, including a retro-aldol cleavage of 3-hydroxy-amino acids, transamination and decarboxylation [7,8]. Although SHMTs from many prokaryotes and eukaryotes have been studied [11][12][13][14][15][16][17], in-depth investigation has focused only on SHMTs from rabbit and sheep liver, as well as E. coli and Bacillus stearothermophilus [6,7,11,18,19].…”

mentioning

confidence: 99%

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

et al. 2009

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The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 m−1·cm−1. The Kd for binding of the enzyme and pyridoxal‐5‐phosphate was 0.14 ± 0.01 μm. An alternative assay for measuring the tetrahydrofolate‐dependent SHMT activity based on the coupled reaction with 5,10‐methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary‐complex mechanism with a kcat value of 0.98 ± 0.06 s−1 and Km values of 0.18 ± 0.03 and 0.14 ± 0.02 mm for l‐serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius’s plot showed a transition temperature of 19 °C. Besides l‐serine, PvSHMT forms an external aldimine complex with d‐serine, l‐alanine, l‐threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate‐independent retro‐aldol cleavage of 3‐hydroxy amino acids. Although l‐serine is a physiological substrate for SHMT in the tetrahydrofolate‐dependent reaction, PvSHMT can also use d‐serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme–quinonoid complex with d‐serine, but not with l‐serine, whereas SHMT from rabbit liver was reported to form an enzyme–quinonoid complex with l‐serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

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“…Data were collected at 277 nm and at a spacing of 0.003 cm with three averages in a continuous scan mode. In the case of the double mutant L85A ⁄ L276A holoenzyme the sedimentation velocity experiments were also performed at 420 nm, aiming to evaluate the presence of PLP covalently bound to the monomeric species as a Schiff base with the active site lysine residue [9]. Sedimentation coefficients and integration of data were obtained using the software sedfit (provided by P. Schuck, National Institutes of Health, Bethesda, MD, USA).…”

Section: Analytical Ultracentrifugation Analysesmentioning

confidence: 99%

The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase

et al. 2008

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Pyridoxal 5′‐phosphate‐dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site‐directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydromethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis.

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Serine hydroxymethyltransferase from Escherichia coli: purification and properties

Cited by 165 publications

References 20 publications

Modeling cellular compartmentation in one‐carbon metabolism

Modeling cellular compartmentation in one‐carbon metabolism

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase

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