Single-turnover Enzyme Research Articles

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.

The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

Oxidative Dearomatization of PLP in Thiamin Pyrimidine Biosynthesis in Candida albicans.

The yeast thiamin pyrimidine synthase THI5p catalyzes one of the most complex organic rearrangements found in primary metabolism. In this reaction, the active site His66 and PLP are converted to thiamin pyrimidine in the presence of Fe(II) and oxygen. The enzyme is a single-turnover enzyme. Here, we report the identification of an oxidatively dearomatized PLP intermediate. We utilize oxygen labeling studies, chemical-rescue-based partial reconstitution experiments, and chemical model studies to support this identification. In addition, we also identify and characterize three shunt products derived from the oxidatively dearomatized PLP.

SNAP-tagging live cells via chelation-assisted copper-catalyzed azide-alkyne cycloaddition.

SNAP-tag is a single-turnover enzyme that has become a powerful tool, hence a popular choice, of targeted cellular protein labeling. Three SNAP-tag substrates that carry the copper-chelating 2-picolyl azide moiety are prepared, one of which has an unconventional 5-pyridylmethyl-substituted guanine structure, rather than the usual benzylguanine that is optimized to be accepted by SNAP-tag. All three substrates are effective in transferring a 2-picolyl azide moiety to SNAP-tag in live cells under conventional labeling conditions (30-minute incubation of cells with labeling reagents at 37 °C under 5% CO2). Live cells that are decorated with chelating azido groups on the extracellular side of membranes undergo copper-catalyzed azide-alkyne cycloaddition (CuAAC) with an ethynyl-functionalized fluorophore to accomplish membrane protein labeling by a fluorescent dye. The chelation-assisted CuAAC labeling step is rapid (<1 minute) with a relatively low dose of the copper catalyst (20 μM), and consequently exerts no ill effect on the labeled cells. A SNAP-tag substrate that carries a non-chelating azide moiety, on the other hand, fails to produce satisfactory labeling under the same constraints. The rapid, live cell-compatible SNAP-tag/chelation-assisted CuAAC two-step method expands the utility of SNAP-tag in protein labeling applications.

Intramolecular electron transfer from biopterin to FeII-O2 complex in nitric oxide synthases occurs at very different rates between bacterial and mammalian enzymes: Direct observation of a catalytically active intermediate

Nitric oxide synthase (NOS) is a cytochrome P450-type mono‑oxygenase that catalyzes the oxidation of L-arginine to nitric oxide. We previously observed that intramolecular electron transfer from biopterin to Fe2+-O2 in Deinococcus radiodurans NOS (DrNOS) using pulse radiolysis. However, the rate of electron transfer in DrNOS (2.2 × 103 s−1) contrasts with a reported corresponding rate (11 s−1) in a mammalian NOS determined using rapid freeze-quench (RFQ) EPR. We applied pulse radiolysis to Bacillus subtilis NOS (bsNOS) and to rat neural NOS oxygenase domain NOS (mNOS). Concurrently, RFQ EPR was used to trap a pterin radical during single-turnover enzyme reactions of the enzymes. By using the pulse radiolysis method, hydrated electrons (eaq−) reduced the heme iron of NOS enzymes. Subsequently, ferrous heme reacted with O2 to form a Fe2+-O2 intermediate. In the presence of pterin, the intermediate of bsNOS was found to convert to other intermediate in the time range of milliseconds. A similar process was determined to have occurred after pulse radiolysis of the pterin-bound mNOS, though the rate was much slower. The intermediates of all of the NOS enzymes further converted to the original ferric form in the time range of seconds. When using the RFQ method, pterin radicals were formed very rapidly in both DrNOS and bsNOS in the time range of milliseconds. In contrast, the pterin radical in mNOS was observed to form slowly, at a rate of ∼20 s−1.

Molecular insights into the endoperoxide formation by Fe(II)/\u03b1-KG-dependent oxygenase NvfI

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.

Cleavage of Influenza RNA Using Artificial RNA‐cleaving Enzyme

Previously, we reported that our designed artificial DNA-cleaving enzyme which comprises our artificial DNA-binding protein and a DNA-cleaving domain inhibited DNA replication of human papilloma virus (HPV) in mammalian cells by cleaving its target HPV ori plasmid sequence-specifically. Our artificial DNA-binding proteins have much higher affinity and selectivity than other DNA-binding proteins such as CRISPR-dCas9. In addition, our artificial DNA-cleaving enzymes cleave target DNA with multiple turnovers while CRISPR acts as a single-turnover enzyme. Thereafter, we applied this methodology for inactivating DNA viruses to RNA viruses. We have recently developed artificial RNA-binding proteins with higher affinity and selectivity than other RNA-binding proteins such as CRISPR-Cas13. In the present study, in order to inhibit RNA replication of influenza virus, we developed artificial RNA-cleaving enzymes which comprise our artificial RNA-binding protein and an RNA-cleaving domain. After we confirmed that an artificial RNA-binding protein specifically bound to its target RNA, we demonstrated that the artificial RNA-cleaving enzymes site-selectively and efficiently cleaved their target RNA sequence in an influenza viral genome in vitro. And we also demonstrated that the artificial RNA-cleaving enzyme effectively inhibited RNA replication of influenza virus in mammalian cells due to cleavage of target RNA. We will present the detail in this conference.

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single ‘priming’ electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2O2‐driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped‐flow spectroscopy, alongside electron paramagnetic resonance and UV‐Vis spectroscopy, we reveal how H2O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.

The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis

Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation 1.Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy 2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling 3– 4.The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355.

Expanding the substrate selectivity of SNAP/CLIP-tagging of intracellular targets.

SNAP-tag belongs to a class of genetic tools of protein labeling that complements fluorescent proteins. This single-turnover enzyme is a mutant of human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT). It accepts, in most cases, label-carrying O6-benzylguanines or benzyl-2-chloro-6-aminopyrimidines as suitable substrates. In this article, strategies and methods to expand the scope of the labels for intracellular proteins of live cells via the actions of SNAP-tag are presented. CLIP-tag is another mutant of the hAGT that was engineered to have mutually exclusive substrate specificity from SNAP-tag. The use of complementary bioorthogonal chemical reactions in conjunction with orthogonal enzymatic SNAP/CLIP-tags for the purpose of dual-color intracellular labeling is also described.

Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single turnover enzyme that displays a stable product state after double-stranded DNA cleavage. Here, we present cryo-EM structures of pre-catalytic, post-catalytic, and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the pre-catalytic state, Cas9 adopts the “checkpoint” conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the post-catalytic state involves a dramatic ~34 Å swing of the HNH domain and disorder of the REC2 recognition domain. The post-catalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the pre-catalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and nucleic acids provide new insights into the mechanism of genome editing by Cas9.

Generic Schemes for Single-Molecule Kinetics. 3: Self-Consistent Pathway Solutions for Nonrenewal Processes.

We recently developed a pathway analysis framework (paper 1) for describing single-molecule kinetics for renewal (i.e., memoryless) processes based on the decomposition of a kinetic scheme into generic structures. In our approach, waiting time distribution functions corresponding to such structures are expressed in terms of self-consistent pathway solutions and concatenated to form measurable probability distribution functions (PDFs), affording a simple way to decompose and recombine a network. Here, we extend this framework to nonrenewal processes, which involve correlations between events, and employ it to formulate waiting time PDFs, including the first-passage time PDF, for a general kinetic network model. Our technique does not require the assumption of Poissonian kinetics, permitting a more general kinetic description than the usual rate approach, with minimal topological restrictiveness. To demonstrate the usefulness of this technique, we provide explicit calculations for our general model, which we adapt to two generic schemes for single-enzyme turnover with conformational interconversion. For each generic scheme, wherein the intermediate state(s) need not undergo Poissonian decay, the functional dependence of the mean first-passage time on the concentration of an external substrate is analyzed. When conformational detailed balance is satisfied, the enzyme turnover rate (related to the mean first-passage time) reduces to the celebrated Michaelis-Menten functional form, consistent with our previous work involving a similar scheme with all rate processes, thereby establishing further generality to this intriguing result. Our framework affords a general and intuitive approach for evaluating measurable waiting time PDFs and their moments, making it a potentially useful kinetic tool for a wide variety of single-molecule processes.

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

An histidine covalent receptor and butenolide complex mediates strigolactone perception.

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D-ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases and is known to hydrolyze the bond between the ABC lactone and the D-ring. Here we characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Coevolution of RtcB and Archease created a multiple-turnover RNA ligase.

RtcB is a noncanonical RNA ligase that joins either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

Induction of the Sugar-Phosphate Stress Response Allows Saccharomyces cerevisiae 2-Methyl-4-Amino-5-Hydroxymethylpyrimidine Phosphate Synthase To Function in Salmonella enterica.

Thiamine pyrophosphate is a required cofactor for all forms of life. The pyrimidine moiety of thiamine, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), is synthesized by different mechanisms in bacteria and plants compared to fungi. In this study, Salmonella enterica was used as a host to probe requirements for activity of the yeast HMP-P synthase, Thi5p. Thi5p synthesizes HMP-P from histidine and pyridoxal-5-phosphate and was reported to use a backbone histidine as the substrate, which would mean that it was a single-turnover enzyme. Heterologous expression of Thi5p did not complement an S. enterica HMP-P auxotroph during growth with glucose as the sole carbon source. Genetic analyses described here showed that Thi5p was activated in S. enterica by alleles of sgrR that induced the sugar-phosphate stress response. Deletion of ptsG (encodes enzyme IICB [EIICB] of the phosphotransferase system [PTS]) also allowed function of Thi5p and required sgrR but not sgrS. This result suggested that the role of sgrS in activation of Thi5p was to decrease PtsG activity. In total, the data herein supported the hypothesis that one mechanism to activate Thi5p in S. enterica grown on minimal medium containing glucose (minimal glucose medium) required decreased PtsG activity and an unidentified gene regulated by SgrR. This work describes a metabolic link between the sugar-phosphate stress response and the yeast thiamine biosynthetic enzyme Thi5p when heterologously expressed in Salmonella enterica during growth on minimal glucose medium. Suppressor analysis (i) identified a mutant class of the regulator SgrR that activate sugar-phosphate stress response constitutively and (ii) determined that Thi5p is conditionally active in S. enterica. These results emphasized the power of genetic systems in model organisms to uncover enzyme function and underlying metabolic network structure.

Parallel versus Off-Pathway Michaelis-Menten Mechanism for Single-Enzyme Kinetics of a Fluctuating Enzyme.

Recent fluorescence spectroscopy measurements of the turnover time distribution of single-enzyme turnover kinetics of β-galactosidase provide evidence of Michaelis-Menten kinetics at low substrate concentration. However, at high substrate concentrations, the dimensionless variance of the turnover time distribution shows systematic deviations from the Michaelis-Menten prediction. This difference is attributed to conformational fluctuations in both the enzyme and the enzyme-substrate complex and to the possibility of both parallel- and off-pathway kinetics. Here, we use the chemical master equation to model the kinetics of a single fluctuating enzyme that can yield a product through either parallel- or off-pathway mechanisms. An exact expression is obtained for the turnover time distribution from which the mean turnover time and randomness parameters are calculated. The parallel- and off-pathway mechanisms yield strikingly different dependences of the mean turnover time and the randomness parameter on the substrate concentration. In the parallel mechanism, the distinct contributions of enzyme and enzyme-substrate fluctuations are clearly discerned from the variation of the randomness parameter with substrate concentration. From these general results, we conclude that an off-pathway mechanism, with substantial enzyme-substrate fluctuations, is needed to rationalize the experimental findings of single-enzyme turnover kinetics of β-galactosidase.

Insights into the phosphoryl transfer catalyzed by cAMP-dependent protein kinase

Protein kinases are involved in a number of cell signaling pathways. They catalyze phosphorylation of proteins and regulate the majority of cellular processes (such as growth, differentiation, lipid metabolism, regulation of sugar, nucleic acid synthesis, etc.). Chemically, protein kinases covalently transfer the gamma-phosphate group of a nucleoside triphosphate (e.g. ATP) to a hydroxyl group of a Ser, Thr or Tyr residue of substrate protein or peptide. The reaction involves moving hydrogen atoms between the enzyme, substrate and nucleoside. The unanswered question is whether the proton transfer from the Ser residue happens before the phosphoryl transfer using the general acid-base catalyst, Asp166, or after the reaction went through the transition state by directly protonating the phosphate group. To address this key question about the phosphoryl transfer, we determined a number of X-ray structures of ternary complexes of catalytic subunit of cAMP-dependent protein kinase (PKAc) with various substrates, nucleotides and cofactors. Importantly, we were able to trap and mimic the initial (Michaelis complex) and final (product complex) stages of the reaction. The results demonstrate that Mg2+, Ca2+, Sr2+, and Ba2+ metal ions bind to the active site and facilitate the reaction to produce ADP and a phosphorylated peptide. The study also revealed that metal-free PKAc can facilitate the phosphoryl transfer reaction; a result that was confirmed with single turnover enzyme kinetics measurements. Comparison of the product and the pseudo-Michaelis complex structures, in conjunction with molecular dynamics simulations, reveals conformational, coordination, and hydrogen bonding changes that help further our understanding of the mechanism, roles of metals, and active site residues involved in PKAc activity.

Metal-free cAMP-dependent protein kinase can catalyze phosphoryl transfer.

X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na+ or K+ coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing 32P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis.

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the "nineteen complex" associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.

Thiamin Pyrimidine Biosynthesis in Candida albicans: A Remarkable Reaction between Histidine and Pyridoxal Phosphate

In Saccharomyces cerevisiae , thiamin pyrimidine is formed from histidine and pyridoxal phosphate (PLP). The origin of all of the pyrimidine atoms has been previously determined using labeling studies and suggests that the pyrimidine is formed using remarkable chemistry that is without chemical or biochemical precedent. Here we report the overexpression of the closely related Candida albicans pyrimidine synthase (THI5p) and the reconstitution and preliminary characterization of the enzymatic activity. A structure of the C. albicans THI5p shows PLP bound at the active site via an imine with Lys62 and His66 in close proximity to the PLP. Our data suggest that His66 of the THI5 protein is the histidine source for pyrimidine formation and that the pyrimidine synthase is a single-turnover enzyme.