Mechanism of N¹⁰‐formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors

Abstract

N 10 -formyltetrahydrofolate synthetase (FTHFS) is a folate enzyme that catalyzes the formylation of tetrahydrofolate (THF) in an ATP dependent manner. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, complexed with (1) a catalytic intermediate-formylphosphate (XPO) and product-ADP; (2) with an inhibitory substrate analog-folate; (3) with XPO and an inhibitory THF analog, ZD9331, were used to analyze the enzyme mechanism. Nucleophilic attack of the formate ion on the gamma phosphate of ATP leads to the formation of XPO and the first product ADP. A channel that leads to the putative formate binding pocket allows for the binding of ATP and formate in random order. Formate binding is due to interactions with the gamma-phosphate moiety of ATP and additionally to two hydrogen bonds from the backbone nitrogen of Ala276 and the side chain of Arg97. Upon ADP dissociation, XPO reorients and moves to the position previously occupied by the beta-phosphate of ATP. Conformational changes that occur due to the XPO presence apparently allow for the recruitment of the third substrate, THF, with its pterin moiety positioned between Phe384 and Trp412. This position overlaps with that of the bound nucleoside, which is consistent with a catalytic mechanism hypothesis that FTHFS works via a sequential ping-pong mechanism. More specifically, a random bi uni uni bi ping-pong ter ter mechanism is proposed. Additionally, the native structure originally reported at a 2.5 Å resolution was redetermined at a 2.2 Å resolution.

Time passes yet errors remain: Comments on the structure of N10‐formyltetrahydrofolate synthetase

“…In a recent Protein Science article, the authors concluded their studies with a proposal for this enzyme's catalytic mechanism. 2 The structures presented in the article reiterated the initially proposed two amino-acid deletion that was proposed nearly 12 years ago to explain the experimental densities derived at that time. Here, we investigated the divergence between the reported sequence in M. thermoacetica N 10 -formyltetrahydrofolate synthetase and the sequence derived from the crystal structure of this enzyme.…”

“…These errors resulted in a cascade of inaccuracies that raised concerns about main findings of this recent article. 2 The two amino-acid deletion was proposed to explain insufficient density to fit the full reported sequence. However, the site of deletion contained highly conserved Ser residue and was located at the beta strand.…”

Early bioenergetic evolution

“…ATP investment (a concept familiar from glycoysis) is required at the endergonic formyl-H 4 F synthetase step, formyl phosphate (figure 2 c ) being the ‘activated’ ATP-generated reaction intermediate, both in E. coli [154,155] and in the acetogenic enzyme [156]. The methyl group of methyl-H 4 F is transferred via CoFeSP to CODH/ACS where the acetyl-CoA is released.…”

SIGN EPISTASIS LIMITS EVOLUTIONARY TRADE-OFFS AT THE CONFLUENCE OF SINGLE- AND MULTI-CARBON METABOLISM INMETHYLOBACTERIUM EXTORQUENSAM1

“…A, B; Celeste et al. ). Without this key intermediate, FtfL 1101 is unlikely to form its final product, formyl‐H 4 F. Enzyme assays confirmed the complete loss of FtfL function due to ftfL 1101 (Fig.…”

“…) using FtfL from Moorella thermoacetica (PDB 4JJZ; 57% amino acid sequence identity) as a template (Celeste et al. ). Alignment of the model to 4JJZA in the presence of ADP and formyl phosphate—as well as model visualization, in silico site‐directed mutagenesis, and figure preparation—were all performed using PyMOL (Schrodinger, LLC ).…”