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Deoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels,Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.

SAMHD1 is a dGTP-activated deoxynucleoside triphosphate triphosphohydrolase (dNTPase) whose dNTPase activity has been linked to HIV/SIV restriction. The mechanism of its dGTP-activated dNTPase function remains unclear. Recent data also indicate that SAMHD1 regulates retrotransposition of LINE-1 elements. Here we report the 1.8-A crystal structure of homotetrameric SAMHD1 in complex with the allosteric activator and substrate dGTP/dATP. The structure indicates the mechanism of dGTP-dependent tetramer formation, which requires the cooperation of three subunits and two dGTP/dATP molecules at each allosteric site. Allosteric dGTP binding induces conformational changes at the active site, allowing a more stable interaction with the substrate and explaining the dGTP-induced SAMHD1 dNTPase activity. Mutations of dGTP binding residues in the allosteric site affect tetramer formation, dNTPase activity and HIV-1 restriction. dGTP-triggered tetramer formation is also important for SAMHD1-mediated LINE-1 regulation. The structural and functional information provided here should facilitate future investigation of SAMHD1 function, including dNTPase activity, LINE-1 modulation and HIV-1 restriction.

The binding of modifiers and substrates to the ribonucleotide reductase from Lactobacillus leichmannii has been studied with the use of enzyme highly purified by a modified preparative procedure. This monomeric enzyme represents a simpler system than classical allosteric enzymes since the study of allosterism, regulation, and conformational responses in the latter is complicated by interaction between subunits. Analysis of earlier kinetic results indicates that ribonucleotide substrates bind weakly to the regulatory site (KD for GTP, 4 m m ) compared with binding to the catalytic site (KD for GTP 0.24 m m ), and in so doing produce substrate activation. The apparent rate constant for product formation when GTP is not bound to the regulatory site is 31% of that observed when GTP occupies the regulatory site. Part of this effect is due to tighter binding of coenzyme when substrate is bound to the regulatory site, a result confirmed by direct measurement of adenosylcobalamin binding. The effect of modifiers such as dGTP which accelerate the reduction of specific substrates is also in part due to increased coenzyme binding as measured both kinetically and by equilibrium dialysis and ultrafiltration methods. The modifiers also seem to have an obligatory role in the enzyme mechanism. Without their presence neither formation of the radical-pair intermediate from adenosylcobalamin nor associated reactions (coenzyme degradation, coenzyme H-exchange with water) occur under the experimental conditions observed. When modifier concentration dependence for these reactions is compared with estimates of equilibrium constants for dissociation of modifiers and substrates from complexes with the regulatory and catalytic sites it appears likely that binding at the regulatory site is responsible. Calculation of coenzyme saturation under the experimental conditions indicates, however, that insignificant amounts of coenzyme are bound to the enzyme except when the regulatory site is occupied, and this effect adequately explains the necessity for modifiers. Binding of dGTP does not alter the circular dichroism of the enzyme in the far-ultraviolet region (200 to 250 nm) but causes significant changes in the aromatic region, and therefore appears to perturb tryptophyl and tyrosyl residues though not necessarily directly. Measurements have been made of the fractional change in sedimentation coefficient due to dGTP binding to the enzymes by the use of an adjustable Raleigh mask and double sector cell in the Beckman model E ultracentrifuge. Values for Δs/ s of −1.14% at 22° and −1.16% at 37° were obtained. The former, after correction for the fractional increase due to the calculated effect of dGTP on the buoyant weight of the protein, gives a value of (Δs/ s corr = −2.84% . If this is due to increase in hydrodynamic volume of the protein alone, it corresponds to an 8.8% increase. If it were entirely due to increased departure from spherical shape it would correspond to a change in axial ratio from 2.9 to 3.2. In the presence of saturating concentrations of ATP and dihydrolipoate and the maximum permissible concentration of a coenzyme analogue Δs/ s was −0.33%, the corrected value being about −1.99% at 37°. These conformational responses are as great as determined for multimeric enzymes such as in the effect of CTP on aspartate transcarbamylase, and are consistent with relay of the regulatory signal over a considerable intersite distance.