Human and Plasmodium serine hydroxymethyltransferases differ in rate-limiting steps and pH-dependent substrate inhibition behavior

Amornwatcharapong, Watcharee; Maenpuen, Somchart; Chitnumsub, P.; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai

doi:10.1016/j.abb.2017.07.017

Cited by 15 publications

(30 citation statements)

References 31 publications

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“…As a first step, the initial velocity of the reaction was measured at different tRNA concentrations, keeping L-serine concentration fixed (10 mM) and varying the THF concentration. Results show a typical THF substrate inhibition (33) (Figure 8A), which became less evident at increasing tRNA concentrations. Fitting of data to Equation (3) (see ‘Materials and Methods’ section), a modified Michaelis–Menten equation that accounts for substrate inhibition, showed that while tRNA increased the K m for THF, it did not affect V max .…”

Section: Resultsmentioning

confidence: 94%

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

Guiducci

Paone

Tramonti

et al. 2019

Nucleic Acids Research

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Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5′untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1–UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.

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Section: Resultsmentioning

confidence: 94%

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

Guiducci

Paone

Tramonti

et al. 2019

Nucleic Acids Research

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“…With both A. thaliana isoforms, k cat increases as pH is increased. In this respect, At SHMT4 is different from the human cytosolic isoform that shows an opposite behavior 32 . This discrepancy in behavior might be due to the different experimental conditions used to characterize the enzymes, which include buffering compounds and salts that impact ionic strength.…”

Section: Resultsmentioning

confidence: 95%

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models

Ruszkowski

Sekula

Ruszkowska

et al. 2019

Sci Rep

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Serine hydroxymethyltransferases (SHMTs) reversibly transform serine into glycine in a reaction accompanied with conversion of tetrahydrofolate (THF) into 5,10-methylene-THF (5,10-meTHF). In vivo, 5,10-meTHF is the main carrier of one-carbon (1C) units, which are utilized for nucleotide biosynthesis and other processes crucial for every living cell, but hyperactivated in overproliferating cells (e.g. cancer tissues). SHMTs are emerging as a promising target for development of new drugs because it appears possible to inhibit growth of cancer cells by cutting off the supply of 5,10-meTHF. Methotrexate (MTX) and pemetrexed (PTX) are two examples of antifolates that have cured many patients over the years but target different enzymes from the folate cycle (mainly dihydrofolate reductase and thymidylate synthase, respectively). Here we show crystal structures of MTX and PTX bound to plant SHMT isozymes from cytosol and mitochondria—human isozymes exist in the same subcellular compartments. We verify inhibition of the studied isozymes by a thorough kinetic analysis. We propose to further exploit antifolate scaffold in development of SHMT inhibitors because it seems likely that especially polyglutamylated PTX inhibits SHMTs in vivo. Structure-based optimization is expected to yield novel antifolates that could potentially be used as chemotherapeutics.

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“…Substrate inhibition was also observed with human SHMT1 and shown to result from the rate-limiting release of glycine, which leads to formation of the enzyme–glycine–THF ternary complex. This phenomenon is strongly dependent on pH . It is not known whether a similar substrate inhibition takes place with l -serine and the substrates involved in the reverse glycine to l -serine reaction (i.e., glycine and CH 2 -THF).…”

mentioning

confidence: 99%

Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties

et al. 2018

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Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of L-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the serine hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our investigation, which allowed the determination of all kinetic parameters of serine hydroxymethyltransferase forward and backward reactions, uncovered a previously unobserved substrate inhibition by L-serine and highlighted several interesting differences between SHMT1 and SHMT2. In particular, SHMT2 maintains a pronounced tetrahydrofolate substrate inhibition even at the alkaline pH characteristic of the mitochondrial matrix, whereas with SHMT1 this is almost abolished. At this pH, SHMT2 also shows a catalytic efficiency that is much higher than that of SHMT1. These observations suggest that such different properties represent an adaptation of the isoforms to the respective cellular environments and that substrate inhibition may be a form of regulation.

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Human and Plasmodium serine hydroxymethyltransferases differ in rate-limiting steps and pH-dependent substrate inhibition behavior

Cited by 15 publications

References 31 publications

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

Structural basis of methotrexate and pemetrexed action on serine hydroxymethyltransferases revealed using plant models

Human Cytosolic and Mitochondrial Serine Hydroxymethyltransferase Isoforms in Comparison: Full Kinetic Characterization and Substrate Inhibition Properties

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