Functional Analysis of Substrate and Cofactor Complex Structures of a Thymidylate Synthase-Complementing Protein

Abstract

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

“…2B) confirming the ability of DXMS data to detect and localize such disordered regions. Interestingly, these regions also appear to be involved in the binding of the enzyme substrate and adopt a structured conformation after binding ligand (45). This finding suggests that DXMS can also provide some localized prediction of substrate and cofactor binding sites.…”

“…The structure of T. maritima thy1 protein TM0449 has been determined to 2.25 Å (45). Its exchange map demonstrated two segments (more than or equal to four residues in each) with rapid exchange, labeled A (Phe-31-Glu-38) and B (Ser-88-Lys-93), and several isolated rapidly exchanging amides in groups of three or less, scattered throughout the sequence (Fig.…”

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

“…Furthermore, dUMP has been cocrystalized with T. maritima and M. tuberculosis ThyX (20) and NADPH was observed in the active site of M. tuberculosis ThyX (27); however, the position of the methylenetetrahydrofolate within the enzyme is not known. Under low-pH conditions (pH 4.5 to 5.5), NADPH was observed to displace FAD from preformed crystals in M. tuberculosis ThyX (26).…”

Configurational fluctuations and flavin-substrate interactions in the flavoenzyme ThyX studied by time- and spectrally resolved fluorescence

“…The spectral shape remains unchanged after ~200 ps and is significantly different from that of FAD in solution, implying that free FAD does not contribute ( fig 3A). According to the crystal structure of ThyX [6] Tyr91 is the closest aromatic residue to the FAD cofactor, and therefore a likely candidate as a main fluorescence quencher. Indeed, in the mutant Y91F where this Tyr was mutated to ET-incapable but sterically similar Phe, fluorescence decay kinetics are much slower compared to WT (fig 2).…”

“…For this reaction the enzyme accepts a methyl group from a (second) folate substrate and electrons from the flavin-cofactor after being pre-reduced by NADPH. In the absence of the other substrates, dUMP tightly binds close to oxidized FAD [6] leading to dramatic quenching of FAD fluorescence (fig 2). We resolved that the main decay occurs as fast as ~200 fs.…”