Primitive Enzyme Research Articles

Spontaneous Emergence of Lipid Vesicles in a Coacervate-Based Compartmentalized System.

The spontaneous emergence of lipid vesicles in the absence of evolved biological machinery represents a major challenge for bottom-up synthetic biology. We show that coacervate microdroplets could create a compartmentalized environment that enriches lipid molecules and facilitates their spontaneous assembly into lipid vesicles. These vesicles can escape from the coacervate microdroplets in a continuous process under non-equilibrium conditions, resembling a constant production process akin to a "primitive enzyme" factory assembly line. These findings significantly extend our understanding of the intricate interaction between lipid molecules and coacervate microdroplets, shedding light on the emergence of cellular systems and offering a new perspective on the conditions necessary for the development of life on Earth.

Spontaneous Emergence of Lipid Vesicles in a Coacervate‐Based Compartmentalized System

The spontaneous emergence of lipid vesicles in the absence of evolved biological machinery represents a major challenge for bottom‐up synthetic biology. We show that coacervate microdroplets could create a compartmentalized environment that enriches lipid molecules and facilitates their spontaneous assembly into lipid vesicles. These vesicles can escape from the coacervate microdroplets in a continuous process under non‐equilibrium conditions, resembling a constant production process akin to a "primitive enzyme" factory assembly line. These findings significantly extend our understanding of the intricate interaction between lipid molecules and coacervate microdroplets, shedding light on the emergence of cellular systems and offering a new perspective on the conditions necessary for the development of life on Earth.

Nickel-organo compounds as potential enzyme precursors under simulated early Earth conditions

The transition from inorganic catalysis through minerals to organic catalysis by enzymes is a necessary step in the emergence of life. Our work is elucidating likely reactions at the earliest moments of Life, prior to the existence of enzymatic catalysis, by exploring essential intersections between nickel bioinorganic chemistry and pterin biochemistry. We used a prebiotically-inspired acetylene-containing volcanic hydrothermal experimental environment to shed light on the efficient formation of nickel-organo complexes. The simplest bis(dithiolene)nickel complex (C2H2S2)2Ni was identified by UV/Vis spectroscopy, mass spectrometry, nuclear magnetic resonance. Its temporal progression and possible function in this simulated early Earth atmosphere were investigated by isolating the main bis(dithiolene)nickel species from the primordial experimental setup. Using this approach, we uncovered a significant diversity of nickel-organo compositions by identifying 156 elemental annotations. The formation of acetaldehyde through the subsequent degradation of these organo-metal complexes is intriguing, as it is reminiscent of the ability of Pelobacter acetylenicus to hydrate acetylene to acetaldehyde via its bis(dithiolene)-containing enzyme acetylene hydratase. As our findings mechanistically characterize the role of nickel sulfide in catalyzing the formation of acetaldehyde, this fundamental pre-metabolic reaction could play the role of a primitive enzyme precursor of the enzymatic acetylene metabolism and further strengthen the role of acetylene in the molecular origin of life.

Temperature-Dependent Kinetic Isotope Effects in R67 Dihydrofolate Reductase from Path-Integral Simulations.

Calculation of temperature-dependent kinetic isotope effects (KIE) in enzymes presents a significant theoretical challenge. Additionally, it is not trivial to identify enzymes with available experimental accurate intrinsic KIEs in a range of temperatures. In the current work, we present a theoretical study of KIEs in the primitive R67 dihydrofolate reductase (DHFR) enzyme and compare with experimental work. The advantage of R67 DHFR is its significantly lower kinetic complexity compared to more evolved DHFR isoforms. We employ mass-perturbation-based path-integral simulations in conjunction with umbrella sampling and a hybrid quantum mechanics–molecular mechanics Hamiltonian. We obtain temperature-dependent KIEs in good agreement with experiments and ascribe the temperature-dependent KIEs primarily to zero-point energy effects. The active site in the primitive enzyme is found to be poorly preorganized, which allows excessive water access to the active site and results in loosely bound reacting ligands.

Primordial chemistry and enzyme evolution in a hot environment

Ever since the publication of Darwin's Origin of Species, questions have been raised about whether enough time has elapsed for living organisms to have reached their present level of complexity by mutation and natural selection. More recently, it has become apparent that life originated very early in Earth's history, and there has been controversy as to whether life originated in a hot or cold environment. This review describes evidence that rising temperature accelerates slow reactions disproportionately, and to a much greater extent than has been generally recognized. Thus, the time that would have been required for primordial chemistry to become established would have been abbreviated profoundly at high temperatures. Moreover, if the catalytic effect of a primitive enzyme (like that of modern enzymes) were to reduce a reaction's heat of activation, then the rate enhancement that it produced would have increased as the surroundings cooled, quite aside from changes arising from mutation (which is itself highly sensitive to temperature). Some nonenzymatic catalysts of slow reactions, including PLP as a catalyst of amino acid decarboxylation, and the Ce(IV) ion as a catalyst of phosphate ester hydrolysis, have been shown to meet that criterion. The work reviewed here suggests that elevated temperatures collapsed the time required for early evolution on Earth, furnishing an appropriate setting for exploring the vast range of chemical possibilities and for the rapid evolution of enzymes from primitive catalysts.

The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase

Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate dihydrofolate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). R67 DHFR is a homotetramer that exhibits numerous characteristics of a primitive enzyme, including promiscuity in binding of substrate and cofactor, formation of nonproductive complexes, and the absence of a conserved acid in its active site. Furthermore, R67's active site is a pore, which is mostly accessible by bulk solvent. This study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one-half of the active site pore using numerous interactions with R67. Also, stacking between the nicotinamide ring of the cofactor and the pteridine ring of the substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the p-aminobenzoylglutamate tail of DHF occur in the other half of the pore because of ion pair switching between symmetry-related K32 residues from two subunits. This computational result is supported by experimental results that the loss of these ion pair interactions (located >13 Å from the center of the pore) by addition of salt or in asymmetric K32M mutants leads to altered enzyme kinetics [Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Hicks, S. N., et al. (2004) J. Biol. Chem. 279, 46995-47002]. The tail movement at the edge of the active site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes formation of the transition state. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF tail angle, accomplished by different elements of the dissimilar protein folds.

The Effect of Electrostatic Shielding on H Tunneling in R67 Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) catalyzes the hydride (H) transfer reaction between NADPH and dihydrofolate, and produces tetrahydrofolate and NADP+. R67 DHFR is a plasmid encoded enzyme, and is considered a “primitive enzyme” due to its genomic, structural, and kinetic properties.[1, 2] Interestingly, kinetic studies of R67 DHFR show an enhancement in H-transfer rate with increasing ionic strength.[3] To evaluate the source of this rate enhancement, the temperature dependency of intrinsic kinetic isotope effects (KIEs) was measured and the nature of the H-transfer step was evaluated at low and high ionic strengths. At high ionic strength, the KIEs were less temperature dependent than at lower ionic strength. These findings were evaluated using a Marcus-like model, which suggests that at higher ionic strength, the donor and acceptor of the hydride were better oriented for H-tunneling than the same system at lower ionic strength. This comparison addresses the level of system preparation that brings the reaction coordinate into a tunneling-ready conformation. While the effect is small, it is statistically significant, as apparent from the comparative data and standard deviations presented in the Supplementary Information (SI – Table S2). These data demonstrate the high sensitivity of the methodology that was developed to study this system (see detailed methods in the SI). The differences in electrostatic potential surface between low and high ionic strengths were calculated, and the theoretical findings add a molecular perspective to the experimental data.

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH.DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.

R67, the Other Dihydrofolate Reductase: Rational Design of an Alternate Active Site Configuration

R67 dihydrofolate reductase (DHFR) bears no sequence or structural homologies with chromosomal DHFRs. The gene for this enzyme produces subunits that are 78 amino acids long, which assemble into a homotetramer possessing 222 symmetry. More recently, a tandem array of four gene copies linked in-frame was constructed, which produces a monomer containing 312 amino acids named Quad3. Asymmetric mutations in Quad3 have also been constructed to probe the role of Q67 and K32 residues in catalysis. This present study mixes and matches mutations to determine if the Q67H mutation, which tightens binding approximately 100-fold to both dihydrofolate (DHF) and NADPH, can help rescue the K32M mutation. While the latter mutation weakens DHF binding over 60-fold, it concurrently increases kcat by a factor of 5. Two Q67H mutations were added to gene copies 1 and 4 in conjunction with the K32M mutation in gene copies 1 and 3. Addition of these Q67H mutations tightens binding 40-fold, and the catalytic efficiency (kcat/Km(DHF)) of the resulting protein is similar to that of Quad3. Since these Q67H mutations can mostly compensate for the K32M lesion, K32 must not be necessary for DHF binding. Another multimutant combines the K32M mutation in gene copies 1 and 3 with the Q67H mutation in all gene copies. This mutant is inhibited by DHF but not NADPH, indicating that NADPH binds only to the wild type half of the pore, while DHF can bind to either the wild type or mutant half of the pore. This inhibition pattern contrasts with the mutant containing only the Q67H substitution in all four gene copies, which is severely inhibited by both NADPH and substrate. Since gene duplication and divergence are evolutionary tools for gaining function, these constructs are a first step toward building preferences for NADPH and DHF in each half of the active site pore of this primitive enzyme.

Prebiotic Inhibitory Activity of Protein-Like Molecules to the Template-Directed Formation of Oligoguanylate from Guanosine 5′-Monophosphate 2-Methylimidazolide on a Polycytidylic Acid Template

Abstract The influence of thermal copolymers of amino acids (TCAA) as a primitive protein enzyme model has been investigated to detect catalytic and/or inhibitory activities on the template-directed formation of oligoguanylate (oligo(G)) from guanosine 5′-monophosphate 2-methylimidazolide (2-MeImpG) on a polycytidylic acid template (poly(C)) (TD reaction). Different amino acids and compositions were used to prepare TCAAs. These TCAAs were characterized by gel filtration HPLC and the composition analysis of amino acids using phenyl isothiocyanate. It was found that TCAA including His (TCAA-His) has inhibitory activity to the TD reaction, in which TCAA-His accelerates the hydrolysis of 2-MeImpG. Other types of TCAAs, including Gly, Ala, Val, Glu, and Asp, also accelerate the formation of P1,P2-bis(5′-guanosyl) diphosphate (G5′ppG) and G5′ppG incorporated oligo(G)s during the TD reaction at high concentrations of TCAAs. These reactions are considered as primitive enzyme models for the hydrolysis of nucleotide monomer and the formation of pyrophosphate-containing oligo(G).

Substrate profiles and expression of caffeoyl coenzyme A and caffeic acid O-methyltransferases in secondary xylem of aspen during seasonal development.

Seasonal expression of caffeoyl-CoA O-methyltransferase (EC 2.1.1.104) was analyzed in aspen developing secondary xylem in parallel with caffeate O-methyltransferase (EC 2.1.1.68). Enzyme activity and mRNA levels for both enzymes peaked in the middle of the growing season. These results strongly suggest that both forms of O-methyltransferase were actively participating in lignin precursor biosynthesis during the growing season. To determine the role of each enzyme form, xylem extracts from two days in the growing season were assayed with four substrates: caffeoyl-CoA, 5-hydroxyferuloyl-CoA, caffeate acid and 5-hydroxyferulic acid. Recombinant forms of caffeoyl-CoA and caffeate O-methyltransferase were also assayed with these substrates. The recombinant enzymes have different substrate specificity with the caffeoyl-CoA O-methyltransferase being essentially specific for CoA ester substrates with a preference for caffeoyl-CoA, while caffeate O-methyltransferase utilized all four substrates with a preference for the free acid forms. We suggest that caffeoyl-CoA O-methyltransferase is likely to be responsible for biosynthesis of lignin precursors in the guaiacyl pathway and may represent a more primitive enzyme form leftover from very early land plant evolution. Caffeate O-methyltransferase is more likely to be responsible for lignin precursor biosynthesis in the syringyl pathway, especially since it can catalyze methylation of 5-hydroxyferuloyl-CoA quite effectively. This latter enzyme form then may be considered a more recently evolved component of the lignin biosynthetic pathways of the evolutionarily advanced plants such as angiosperms.

A Primitive Enzyme for a Primitive Cell: The Protease Required for Excystation of Giardia

Protozoan parasites of the genus Giardia are one of the earliest lineages of eukaryotic cells. To initiate infection, trophozoites emerge from a cyst in the host. Excystation is blocked by specific cysteine protease inhibitors. Using a biotinylated inhibitor, the target protease was identified and its corresponding gene cloned. The protease was localized to vesicles that release their contents just prior to excystation. The Giardia protease is the earliest known branch of the cathepsin B family. Its phylogeny confirms that the cathepsin B lineage evolved in primitive eukaryotic cells, prior to the divergence of plant and animal kingdoms, and underscores the diversity of cellular functions that this enzyme family facilitates.

Reverse transcription of the Mauriceville plasmid of Neurospora. Lack of ribonuclease H activity associated with the reverse transcriptase and possible use of mitochondrial ribonuclease H.

The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major plasmid transcript beginning directly opposite the 3' end of the template RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704). Here, we show that the Mauriceville plasmid reverse transcriptase has no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous reverse transcriptase in purified ribonucleoprotein particles remain in a full-length duplex with the template RNA. The column-purified Mauriceville plasmid reverse transcriptase initiates cDNA synthesis by using short DNA primers, which remain attached to the 5' end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Saldanha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We find that these primer DNAs can be precisely removed by S1 nuclease digestion of the initial cDNA.RNA duplex, suggesting a mechanism whereby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H activity, which is present in mitochondrial ribonucleoprotein particle preparations prior to their purification. This mitochondrial RNase H can degrade the endogenous plasmid transcript in ribonucleoprotein particles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to be a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired their RNase H domains from a gene encoding a cellular RNase H.

The Synthesis and Properties of Block-Copoly(amino acid)s with Folded Structures: A Primitive Enzyme Model

As a model of a primitive enzyme block-copoly(amino acid)s con taining L-serine and L-aspartic acid as the repeat unit were prepared. Spectro scopic measurements indicate that some block-copoly(amino acid)s, especially ABAB and ABABA types, do form folded conformations. Hydrolysis reactions of p-nitrophenyl p-guanidinobenzoate were accelerated in the presence of ABAB and ABABA type block-copoly(amino acid)s compared with a random copoly (amino acid). From these results we speculate that primitive enzymes were born by assembling block-copoly(amino acid)s.

Biochemical characterization of equine herpesvirus type 3-induced deoxythymidine kinase purified from lytically infected horse embryo dermal fibroblasts.

Infection of horse KyED cells with equine herpesvirus type 3 (EHV-3) resulted in a sevenfold increase in cytosol deoxythymidine kinase (dTK) activity. The EHV-3 dTK was purified from KyED cytosol dTK by affinity chromatography on deoxythymidine-Sepharose and characterized with respect to its electrophoretic mobility, molecular weight, substrate specificity, phosphate donor specificity, and immunological specificity. The purified EHV-3 dTK migrated in polyacrylamide gels with an Rf of 0.30 and sedimented in glycerol gradients with an S value of 5.13, corresponding to a molecular weight of 83,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single band with a molecular weight of 38,000 to 40,000. Antiserum prepared against the EHV-3 dTK induced in KyED cells neutralized the EHV-3-induced enzyme activity but not the dTK purified from uninfected cells. EHV-3 dTK was less sensitive to feedback inhibition to dTTP and had a lower Ki for the antiviral compound 1-beta-D-arabinofuranyosylthymine and a lower Km for the substrate deoxythymidine. These results indicate that infection of cells with EHV-3 results in the induction of a new virus-coded dTK activity which meets the criteria of Jensen for an evolutionary primitive enzyme.

Deoxythymidine kinase metabolism in equine herpesvirus type 3 infected horse embryo dermal fibroblasts

Noninfected and equine herpesvirus type 3 (EHV-3) infected horse embryo dermal fibroblasts (KyED cells) were examined for deoxythymidine kinase (dTK) activity. The activity of extracts from EHV-3-infected KyED cells was approximately seven-fold greater than that of sham-infected cells. Antibody elicited against disrupted EHV-3 infected KyED cells neutralized the EHV-3-induced enzyme activity but not the dTKs present in extracts from uninfected cells. EHV-3 induction of dTK was confirmed by the demonstration of the inability of the virus strain to replicate in the presence of arabinosylthymine (AraT) in cells which themselves were resistant to the drug. In addition, the induced dTK in EHV-3 infected cells was resistant to feedback inhibition by dTTP, but was sensitive to AraT when tested in vitro. This contrasts with the AraT-resistant, dTTP-sensitive enzyme activity observed in extracts of uninfected cells. Electrophoretic analysis of cell extracts in polyacrylamide gels indicated that the EHV-3-induced enzyme comigrates with the host cell cytosol enzyme. These results indicate that infection of cells with EHV-3 results in the induction of a new dTK activity which appears to meet R. A. Jensen's ( Annu. Rev. Microbiol. 30, 409–425, 1976) criteria for an evolutionary primitive enzyme.

Catalytic activities of thermally prepared poly-alpha-amino acids: effect of aging.

Thermally prepared poly-alpha-amino acids were tested after being stored in the dry state for 5 to 10 years. Polymers effective in catalyzing the hydrolysis of p-nitrophenyl acetate showed the same levels of activity as observed 10 years earlier. Polymers effective in catalyzing the decarboxylation of oxaloacetic acid had in 5 years become insoluble in assay medium; their activity, however, had increased by 32 to 145 percent. The results suggest that particular primitive enzyme molecules could have been stable enough to have contributed to evolutionary processes long after they had been produced.