Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

Magnesium ions play a critical role in catalysis by many enzymes and they contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ions in each step in catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. Increasing Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold by largely increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV RT binds only two metal ions prior to incorporation of a correct base-pair. MD simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. MD simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions the specificity of DNA polymerases.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd ≈ 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide.

Donut PCR: a rapid, portable, multiplexed, and quantitative DNA detection platform with single-nucleotide specificity

Current platforms for molecular analysis of DNA markers are either limited in multiplexing (qPCR, isothermal amplification), turnaround time (microarrays, NGS), quantitation accuracy (isothermal amplification, microarray, nanopore sequencing), or specificity against single-nucleotide differences (microarrays, nanopore sequencing). Here, we present the Donut PCR platform that features high multiplexing, rapid turnaround times, single nucleotide discrimination, and precise quantitation of DNA targets in a portable, affordable, and battery-powered instrument using closed consumables that minimize contamination. We built a bread-board instrument prototype and three assays/chips to demonstrate the capabilities of Donut PCR: (1) a 9-plex mammal identification panel, (2) a 15-plex bacterial identification panel, and (3) a 30-plex human SNP genotyping assay. The limit of detection of the platform is under 10 genomic copies in under 30 minutes, and the quantitative dynamic range is at least 4 logs. We envision that this platform would be useful for a variety of applications where rapid and highly multiplexed nucleic acid detection is needed at the point of care.