The crystal structure of P. falciparum SHMT revealed snapshots of an intriguing disulfide/sulfhydryl switch controlling the functional activity.

Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a hydroxymethyl group from L-serine to tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Our previous investigations have shown that SHMTs from Plasmodium spp. (P. falciparum, Pf; P. vivax, Pv) are different from the enzyme from rabbit liver in that Plasmodium SHMT can use D-serine as a substrate. In this report, the biochemical and biophysical properties of the Plasmodium and the human cytosolic form (hcSHMT) enzymes including ligand binding and kinetics were investigated. The data indicate that, similar to Plasmodium enzymes, hcSHMT can use D-serine as a substrate. However, hcSHMT displays many properties that are different from those of the Plasmodium enzymes. The molar absorption coefficient of hcSHMT-bound pyridoxal-5 0 -phosphate (PLP) is much greater than PvSHMT-bound or PfSHMT-bound PLP. The binding interactions of hcSHMT and Plasmodium SHMT with D-serine are different, as only the Plasmodium enzyme undergoes formation of a quinonoid-like species upon binding to D-serine. Furthermore, it has been noted that hcSHMT displays strong substrate inhibition by tetrahydrofolate (THF) (at THF > 40 lM), compared with SHMTs from Plasmodium and other species. The pH-activity profile of hcSHMT shows higher activities at lower pH values corresponding to a pK a value of 7.8 AE 0.1. Thiosemicarbazide reacts with hcSHMT following a one-step model [k 1 of 12 AE 0.6 M À1 Ás À1 and k À1 of (1.0 AE 0.6) 9 10 À3 s À1 ], while the same reaction with PfSHMT involves at least three steps. All data indicated that the ligand binding environment of SHMT from human and Plasmodium are different, indicating that it should be possible to develop species-selective inhibitors in future studies.

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With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Å resolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.

Background: Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from L-serine and tetrahydrofolate. Results: Results indicate that PvSHMT can bind to either substrate first. The rate constant of glycine formation is similar to k cat . Conclusion: PvSHMT reaction occurs via a random-order mechanism and glycine formation is the rate-limiting step. Significance: The data are useful for future investigation on inhibition of SHMT for antimalarial drug development.