Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Maenpuen, Somchart; Amornwatcharapong, Watcharee; Krasatong, Pasupat; Sucharitakul, Jeerus; Palfey, Bruce A.; Yuthavong, Yongyuth; Chitnumsub, P.; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai

doi:10.1074/jbc.m114.612275

Cited by 11 publications

(11 citation statements)

References 34 publications

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“…The presence of the flexible loop to control oligomerization, cofactor and substrate binding, and product release is crucial for the protein evolution point of view. Within the SHMT family, the lack of the flap motif results in a dimeric structure for SHMTs in prokaryotes such as Escherichia coli (33) and Bacillus stearothermophilus (34) and some eukaryotes such as P. vivax and P. falciparum (3,7,26,28,29), whereas in higher eukaryotes like human and rabbit the structure is tetrameric with the presence of flap (27,30). In other enzyme families, a flexible loop/flap region is present in the Plasmodium bifunctional DHFR-TS enzyme from a unique insert (so-called Insert 1) and stabilizes the domain attachment between DHFR and TS in the homodimeric structure of the enzyme (43).…”

Section: Discussionmentioning

confidence: 99%

“…Conversely, substrate inhibition by THF in the reactions of the Plasmodium enzymes is pH-independent and requires much higher concentrations for inhibition (Ͼ0.4 mM) (7,10,26). Previous studies on the burst kinetics of the reaction indicated that the rate-limiting step of the overall reaction of hcSHMT is glycine product release (10), whereas for the overall reaction of the Plasmodium enzyme, PvSHMT, the rate-limiting step is glycine formation (3).…”

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confidence: 99%

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A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

Ubonprasert

Jaroensuk

Pornthanakasem

et al. 2019

Journal of Biological Chemistry

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Edited by Ruma BanerjeeHuman cytosolic serine hydroxymethyltransferase (hcSHMT) is a promising target for anticancer chemotherapy and contains a flexible "flap motif" whose function is yet unknown. Here, using size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering (SAXS), molecular dynamics (MD) simulations, and ligand-binding and enzyme-kinetic analyses, we studied the functional roles of the flap motif by comparing WT hcSHMT with a flap-deleted variant (hcSHMT/⌬flap). We found that deletion of the flap results in a mixture of apo-dimers and holo-tetramers, whereas the WT was mostly in the tetrameric form. MD simulations indicated that the flap stabilizes structural compactness and thereby enhances oligomerization. The hcSHMT/⌬flap variant exhibited different catalytic properties in (6S)-tetrahydrofolate (THF)-dependent reactions compared with the WT but had similar activity in THF-independent aldol cleavage of ␤-hydroxyamino acid. hcSHMT/⌬flap was less sensitive to THF inhibition than the WT (K i of 0.65 and 0.27 mM THF at pH 7.5, respectively), and the THF dissociation constant of the WT was also 3-fold lower than that of hcSHMT/⌬flap, indicating that the flap is important for THF binding. hcSHMT/ ⌬flap did not display the burst kinetics observed in the WT. These results indicate that, upon removal of the flap, product release is no longer the rate-limiting step, implying that the flap is important for controlling product release. The findings reported here improve our understanding of the functional roles of the flap motif in hcSHMT and provide fundamental insight into how a flexible loop can be involved in controlling the enzymatic reactions of hcSHMT and other enzymes.Serine hydroxymethyltransferase (SHMT 2 ; EC 2.1.2.1.) is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme in which the PLP cofactor facilitates the reversible transfer of a hydroxymethyl group from L-serine to (6S)-tetrahydrofolate (THF) to yield glycine and 5,10-methylenetetrahydrofolate (5,10-CH 2 -THF) as products (1-3). SHMT is one of the enzymes in the deoxythymidylate (dTMP) synthesis cycle in which the other two enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR), also take part in the recycling of folate compounds and production of 5,10-CH 2 -THF via the SHMT reaction. 5,10-CH 2 -THF serves as a methyl donor for the TS reaction to convert dUMP to dTMP, which is a requisite precursor for DNA biosynthesis (4). Due to its important role in DNA biosynthesis, cell proliferation, and cell survival, SHMT is one of the attractive targets for antimalarial (5-12) and anticancer chemotherapy (10,(13)(14)(15)(16).

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

Ubonprasert

Jaroensuk

Pornthanakasem

et al. 2019

Journal of Biological Chemistry

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“…5a,b), indicating that the GlyA function in pathway Ser3 is necessary for Ser biosynthesis, and that Ser is generated from Gly under these conditions. As the GlyA reaction has been reported to function for the generation of Gly from Ser in a number of organisms404142, we examined whether this is also the case in T. kodakarensis . The glyA gene disruption strain was grown in a synthetic amino acid medium depleted of Gly but with Ser.…”

Section: Resultsmentioning

confidence: 96%

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

et al. 2016

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Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization.

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“…Hp SHMT Ile 178 (Val in Ec SHMT) does not favour the same conformer of Phe 171 in Ec SHMT, which in turn is not compatible with a “closed lid” conformation of loop 3 as observed in Ec SHMT ( Fig 5C ). It was previously shown that SHMTs could bind either PLP or tetrahydrofolate substrates independently [61]. In this context, binding of the tetrahydrofolate moiety in Hp SHMT would likely modify the homodimer interface to bridge loop 1 and the required “closed lid” conformer of loop 3, therefore together promoting PLP binding.…”

Section: Resultsmentioning

confidence: 99%

Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori

et al. 2018

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Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a ubiquitous pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes the formation of glycine from serine. The thereby generated 5,10-methylene tetrahydrofolate (MTHF) is a major source of cellular one-carbon units and a key intermediate in thymidylate biosynthesis. While in virtually all eukaryotic and many bacterial systems thymidylate synthase ThyA, SHMT and dihydrofolate reductase (DHFR) are part of the thymidylate/folate cycle, the situation is different in organisms using flavin-dependent thymidylate synthase ThyX. Here the distinct catalytic reaction directly produces tetrahydrofolate (THF) and consequently in most ThyX-containing organisms, DHFR is absent. While the resulting influence on the folate metabolism of ThyX-containing bacteria is not fully understood, the presence of ThyX may provide growth benefits under conditions where the level of reduced folate derivatives is compromised. Interestingly, the third key enzyme implicated in generation of MTHF, serine hydroxymethyltransferase (SHMT), has a universal phylogenetic distribution, but remains understudied in ThyX-containg bacteria. To obtain functional insight into these ThyX-dependent thymidylate/folate cycles, we characterized the predicted SHMT from the ThyX-containing bacterium Helicobacter pylori. Serine hydroxymethyltransferase activity was confirmed by functional genetic complementation of a glyA-inactivated E. coli strain. A H. pylori ΔglyA strain was obtained, but exhibited markedly slowed growth and had lost the virulence factor CagA. Biochemical and spectroscopic evidence indicated formation of a characteristic enzyme-PLP-glycine-folate complex and revealed unexpectedly weak binding affinity of PLP. The three-dimensional structure of the H. pylori SHMT apoprotein was determined at 2.8Ǻ resolution, suggesting a structural basis for the low affinity of the enzyme for its cofactor. Stabilization of the proposed inactive configuration using small molecules has potential to provide a specific way for inhibiting HpSHMT.

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Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Cited by 11 publications

References 34 publications

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

A flap motif in human serine hydroxymethyltransferase is important for structural stabilization, ligand binding, and control of product release

An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori

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