Biochemistry

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. The enzyme that catalyzes this reaction, lipoyl synthase (LipA in bacteria, and LIAS in humans), is a member of the radical S-adenosylmethionine (SAM) superfamily. As such, it requires a [4Fe-4S] cluster cofactor to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA*) that abstract the C6 and C8 hydrogen atoms (H*) of the substrate in two distinct steps. LipAs also contain a second [4Fe-4S] cluster, termed the auxiliary cluster, degraded during turnover as the source of the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX4CX5C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R306SS308Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in a protein that is essentially inactive due to poor substrate binding and positioning in the active site. Multiple different substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which gave greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl peptide, an intermediate in the reaction, and the formation of a desaturated product, determined to be a 6-octenoyl group attached to the peptide substrate. Furthermore, the 3Fe cluster formed during cannibalization of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.

Post-translational modifications usually occur under enzymatic control as a mechanism for regulation of protein activity or localization, but can also occur spontaneously during protein aging and degradation. In contrast, there are few examples of spontaneous post-translational modifications with a significant role in the stability or biochemical function of a bacterial protein. Here we show that a spontaneous post-translational modification is structurally encoded and functionally important in a bacterial solute-binding protein. We show that the solute-binding protein SAR11_0655 from the abundant SAR11 marine bacterium Pelagibacter ubiqueversans contains an unusually stable succinimide residue important for its function in high-affinity binding of the amino acid derivative 5-oxoproline. Spontaneous cyclization of Asn269 to form a succinimide residue was inferred from high-resolution X-ray crystallography and confirmed by liquid chromatography-tandem mass spectrometry. Loss of the succinimide modification via the substitution N269A resulted in a >20 {degrees}C decrease in thermostability and a 15 to 50-fold decrease in binding affinity for 5-oxoproline. Analysis of sequence conservation indicated that both Asn269 and Asp269 are common among SAR11_0655 homologs, and the N269D variant of SAR11_0655 also showed partial formation of succinimide. These results reveal a previously unrecognized mechanism by which proteins exploit spontaneous post-translational chemistry as a design feature to enhance stability and function, and provides a potential basis for identification and design of structurally encoded succinimide residues for engineering of protein thermostability.

In bacteria, the ring-shaped sliding clamp, DnaN, is an essential component of the replication machinery. The clamp encircles the parental DNA strand during replication and binds DNA polymerases and other replication and repair proteins, helping to tether them at their site of action on the DNA strand. These binding partners interact with the clamp via short pentapeptide or hexapeptide sequences known as clamp-binding motifs (CBMs). Although conserved CBM sequences have been identified across different bacterial species, most studies of clamp binding have been performed in the model gram-negative bacterium Escherichia coli and less is known about clamp binding in other bacterial species. In this study, we investigate clamp binding in the model gram-positive bacterium Bacillus subtilis. We use fluorescence polarization binding assays to quantify binding of a range of CBM peptides to the clamps of both E. coli and B. subtilis. We identify similarities in clamp binding between the two species, including similar importance of different amino acids within the conserved pentapeptide motif. However, our results also reveal differences in clamp binding between the two species. Most notably, we find that, although pentapeptide CBMs bind the E. coli and B. subtilis clamps with similar affinity, hexapeptide CBMs bind an order of magnitude more weakly to the B. subtilis clamp. Our results provide new insight into clamp binding in bacteria and point to possible species-specific differences in this essential interaction.

Nearly all carbon on Earth today is fixed by the enzyme ribulose-1,5-bisphopshate carboxylase/oxygenase ( rubisco), which converts carbon dioxide (CO2) to sugar phosphates. All rubiscos measured thus far display a kinetic isotope effect (KIE) where 12CO2 is fixed at a faster rate than 13CO2. The relationship between rubiscos KIE and the carbon isotope composition of plants, algae, and organic matter is central to many fields in the Earth sciences, plant biology, and biochemistry. Currently, all applications assume that the KIE does not vary with temperature. Here, we examine this assumption experimentally with in vitro KIE measurements of two rubiscos from phylogenetically distinct host organisms and rubisco protein clades - a Form I rubisco from the plant, Spinacia oleracea (spinach) and a Form II rubisco from the bacterium Rhodosprillium rubrum. We that find that both KIEs decrease linearly by [~]4.5{per thousand} from 10-35{degrees}C with statistically indistinguishable slopes. We place these results into biological and geologic contexts by comparing them to observed variations in the carbon isotope composition of modern terrestrial plants and marine organic carbon, the geologic carbon isotope record, and rubiscos biochemistry. We show that the measured temperature dependencies are sufficiently large to impact our interpretations of the enzymatic processes that drive variations in rubisco KIEs, as well as applications of stable carbon isotopes in the Earth and biological sciences. Significance StatementThe carbon isotope composition of plants, algae, and organic matter are interpreted with models that assume the kinetic isotope effect of the carbon-fixing enzyme rubisco is temperature-independent, even though temperature varies by tens of degrees across the Earth today and in the past. Here, we demonstrate that the kinetic isotope effect of rubisco is temperature-dependent, suggesting that some of this isotopic variation may be due to intrinsic enzyme properties alone. In addition, though the rubiscos we measured are from diverse organisms (plant vs. bacteria), their KIEs show statistically indistinguishable temperature dependencies. This data forms the basis for future thermodynamic models on rubisco biochemistry.

Membrane-bound decarboxylases couple carboxylic acid decarboxylation to the transport of Na+ ions out of prokaryotic cells. The molecular mechanism of decarboxylase action is not yet known, which contrasts with the progress achieved in studying other primary ion pumps. Measuring decarboxylase activity is complicated by slow keto-enol tautomerization of the substrates during the assay. We found that HEPES exhibits anomalously high efficiency as a general acid catalyst for C-H bond formation during the enol-to-ketone conversion of oxaloacetate. Accordingly, the addition of HEPES to the assay medium eliminated the contribution of tautomerization rate to measured decarboxylation rate. Using the dependence of oxaloacetate tautomerization rate and equilibrium on solvent properties and pH, we established that only the keto form of oxaloacetate is converted by Vibrio cholerae oxaloacetate decarboxylase. Steady-state kinetic measurements did not reveal cooperativity in oxaloacetate conversion and Na+ binding. The effects of ionophores (CCCP, valinomycin, and ETH157) on proton transport in pyranine-loaded membrane vesicles prepared from V. cholerae cells indicated that the proton required for the conversion of oxaloacetate to pyruvate is taken up from the cytoplasmic side of the membrane. Furthermore, the effects suggested that {Delta}pH generation is caused by secondary electrophoretic proton transport in exchange for Na+.These findings advance our understanding of the molecular mechanism of the decarboxylation-supported Na+ transport in bacteria.

Natural product synthesis by non-ribosomal peptide synthetases (NRPS) is greatly defined by the substrate selectivity of the adenylation (A) domains. Previous assays for specificity determination were mainly performed in vitro and were requiring protein purification. In this work, we developed - based on NRPS engineering - a novel in vivo assay suitable for high-throughput application named ASCR (A domain screening). Using the recently described XUT fusion sites, A domains and their upstream condensation domains were assembled as di-domains to characterized NRPS model system, which allowed detection of defined tripeptide products via mass spectrometry directly after cell culture extraction. We evaluated the assay by screening in total 54 A domains from five known and seven uncharacterized NRPS, covering a broad range organism taxonomy and GC content of the investigated NRPS-encoding genes. Additionally, we applied the assay to elucidate and confirm the structures of novel cyclic pentapeptides derived from three novel NRPS from Photorhabdus temperata K122.

tRNA wobble base uridines are heavily modified to influence anticodon:codon base pairing and tune the structure of the anticodon stem loop for efficient and accurate translation. Both Gram-positive and negative bacteria, as well as certain archaea, modify wobble uridines to contain either a 5-carboxymethylaminomethyl (cmnm5) or a 5-methylaminomethyl (mnm5) moiety, as well as in some instances a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further modified in some tRNAs to mnm5U. The steps to synthesize the latter are catalyzed by non-orthologous enzymes in distantly related bacteria. Escherichia coli utilizes a single bifunctional enzyme, MnmC, to both demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U, while Bacillus subtilis relies on the radical SAM (rSAM) enzyme MnmL, followed by the stand-alone MnmM methylase for mnm5U production. It was previously noted that E. coli and related bacteria that encode MnmC can also contain homologs of MnmL. As an E. coli mnmC mutant accumulates cmnm5U, the function of the MnmL homolog in this organism, YhcC, was unknown. Here, we find YhcC is necessary for cmnm5s2U demodification in vivo under anaerobic growth, and that the same MnmC-mediated demodification activity requires O2 and only occurs under aerobic growth. In vitro reaction experiments demonstrate that purified YhcC, reconstituted to its [4Fe-4S] form, is able to bind tRNA and catalyze the nm5s2U-tRNA synthesis from cmnm5s2U-tRNA. Together, these results define the heretofore unknown biochemistry of the E. coli rSAM enzyme YhcC and show this enzyme replaces MnmC under anaerobic conditions to carry out the synthesis of nm5s2U. These parallel tRNA modification pathways highlight how E. coli has adapted to maintain biosynthesis of a critical wobble base modification under both aerobic and anaerobic growth. General audience summaryTransfer RNA (tRNA) molecules serve as critical components in protein synthesis through their direct interaction with the ribosome and messenger RNA (mRNA). During protein synthesis, tRNA molecules utilize an anticodon composed of three bases that recognize a complementary mRNA codon. At the 3CCA end of tRNA the amino acid corresponding to the anticodon sequence is incorporated into the growing polypeptide chain. While canonical Watson-Crick base pairing, pairing between U:A and G:C, occurs between the second and third bases of the anticodon and the corresponding bases of the codon, there is increased flexibility in the ribosome at the first position of the anticodon. This results in non-canonical base pairing and allows a single tRNA molecule to recognize multiple codons. Modification at this site restricts or enhances this "wobble" base pairing. Bacteria often install mnm5s2U34 to various tRNAs to facilitate proper and efficient translation. In E. coli, three proteins are responsible for this hypermodification pathway: MnmE and MnmG convert s2U to cmnm5s2U, while MnmC subsequently removes the carboxymethyl group to generate nm5s2U and methylates this intermediate into the ultimate product, mnm5s2U. We found that an additional enzyme, YhcC, is required for mnm5s2U synthesis in anaerobic conditions, specifically at the cmnm5s2U demodification step. Previous studies showed that MnmC-catalyzed demodification is dependent on FAD for the initial oxidation of tRNA substrate, generating FADH2. We propose that FADH2 recycling is dependent on O2 to regenerate FAD, allowing multiple catalytic turnovers. We show that, in vitro, O2 is required for cmnm5s2U demodification by MnmC, supporting a new model of mnm5s2U synthesis with alternative aerobic and anaerobic routes. Conservation of multiple enzymes that perform similar chemistry, albeit under differing environmental conditions, highlights the importance of maintaining wobble base modifications as well as the ability of bacteria to adapt to their surroundings.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.

Acetyl-coenzyme A (CoA) is a central metabolic intermediate that links carbon and energy metabolism across all domains of life. The conversion of acetate and acetyl-CoA is carried out by three enzyme pathways: acetate kinase/phosphotransacetylase, ADP-forming acetyl-CoA synthetase, and AMP-forming acetyl-CoA synthetase (Acs). Acs enzymes serve critical physiological roles across diverse organisms generally by catalyzing a reversible two-step reaction forming acetyl-CoA and AMP from acetate and ATP. Isolated from the wastewater reclamation facility in Norman, Oklahoma, Syntrophus aciditrophicus strain SB (Sa) relies on an AMP-forming acetyl-CoA synthetase (SaAcs1) that favors synthesizing acetate and ATP from acetyl-CoA and AMP, in contrast to all previously characterized Acs enzymes. The origin of this preference and the structural determinants of both the thioester-forming step and catalytic directionality remain poorly understood. Here, we report a 2.2 [A] crystal structure of full-length SaAcs1 in the adenylation conformation with acetyl-AMP bound in the active site. Structural comparison to the extensively characterized Acs enzymes from Salmonella enterica (SeAcs) and Cryptococcus neoformans (CnAcs) revealed a displaced CoA-binding loop in SaAcs1. Enzymatic assays confirmed that SaAcs1 preferentially catalyzes the ATP-forming reaction. Site-directed mutagenesis demonstrated that reversion of two residues, G196 and T197, at the beginning of the CoA-binding loop to the consensus sequence repositions the loop and shifts catalytic preference toward the AMP-forming direction. Together, these results establish the CoA-binding loop and G196 and T197 as the primary structural determinants of directional preference in SaAcs1.

Natural product (NPs) and their derivatives are a major source of small-molecule drugs, and the building blocks of these NPs are often amino acids. These include both proteinogenic and nonproteinogenic amino acids (NPAAs), the latter of which expand the structural diversity of NPs. Homologation, or the addition of a methylene group to the amino acid side chain, is one modification that generates NPAAs. If the natural homologation pathway can be characterized and engineered, it could be used to diversify NPs. In this study, we investigated the terminal enzyme of this pathway, HphB, to determine its substrate scope. HphB was tested with various substrates that differed in backbone and/or side chain structures relative to its natural substrate. The results showed that HphB exhibits high promiscuity toward substrates with different side chains while maintaining strict specificity for the substrate backbone. Comparative analysis with two homologous enzymes from primary metabolic pathways revealed that HphB displays markedly higher substrate promiscuity. Bioinformatics analysis and structural modeling suggest that this promiscuity arises from the absence of a "lid" over the active site, resulting in increased solvent exposure of the substrate side chain. This study highlights the unique substrate flexibility of HphB and is a step toward engineering the homologation pathway to generate amino acid derivatives.

Caulobacter crescentus is a gram-negative bacterium that produces the anionic sphingolipid ceramide diphosphoglycerate that can substitute for the lipopolysaccharide component of the outer membrane. ccna_01210 is a gene in the operon for ceramide diphosphoglycerate synthesis and encodes for the enzyme, CpgD. CpgD is a magnesium-dependent CTP:phosphoglycerate cytidylyltransferase that catalyzes the synthesis of CDP-glycerate, and a pyrophosphate byproduct. CpgD displays substrate specificity for the nucleotide CTP and a preference for 2-phospho-D-glycerate over other phosphoglycerate enantiomers and isomers. Here, we present five high resolution structures for CpgD in various catalytic states that rationalize the specificity and preference of CpgD for its two substrates. This includes structures of apo CpgD, a CpgD-CTP-Mg2+ ternary complex, and three CpgD-CDP-glycerate-pyrophosphate-Mg2+ product bound complexes. The structures reveal CpgD nucleotide specificity is mediated by favorable hydrogen bonding interactions with the cytosine nucleobase of CTP, while the preference for 2-phospho-D-glycerate occurs due to favorable van der Waals interactions with the 2D enantiomer and unfavorable steric clashes with the 2L enantiomer. A catalytic mechanism involving a pentacoordinate transition state is proposed based on the observed stereochemical inversion of the -phosphate in the substrate CTP in comparison to the -phosphate of the product CDP-glycerate. Overall, this provides insights into the catalytic mechanism, nucleotide specificity, and enantiomeric substrate preference of the cytidylyltransferase CpgD that participates in a unique pathway of bacterial sphingolipid synthesis.

Codon optimality promotes efficient translation and, as recent research has shown, also extends mRNA lifetimes. However, how control is distributed between translation and mRNA degradation remains unclear. We show that this relative impact depends strongly on the measurement approach. Using fluorescent protein reporters can underestimate codon-optimality-dependent increases in translation efficiency. Conversely, analyses based on poly(A)-selected RNA overestimate the impact on translation, because stable transcripts undergoing poly(A) shortening are often inefficiently captured, leading to skewed protein-to-mRNA ratios. This technical bias is not offset by the marginal decline in ribosomal association observed as mRNAs age. Estimates based on total RNA measurements redistribute some of the control attributed to translation to mRNA stability, making the contributions comparable for mRNAs with shorter coding sequences. For longer mRNAs, codon optimality increasingly controls elongation speed, with a greater effect on translation efficiency than on degradation. These insights highlight the importance of measurement strategy for accurately quantifying the determinants of mRNA stability and protein synthesis.

Selective protein degradation by the ubiquitin-proteasome system frequently involves substrate recognition via short linear motifs known as degradation signals or degrons. Whereas degrons located at protein N-termini (N-degrons) have been extensively studied, our understanding of C-terminal degrons (C-degrons) is comparatively limited. Previously, we showed that the yeast SCF ubiquitin ligase and one of its substrate receptor subunits, the F-box protein Das1, target a broad range of C-degrons and implicated SCFDas1 in orphan quality control. Here, we sought to determine how Das1 recognizes its substrates. By combining in vivo competition assays with structural modeling and mutational analysis, we demonstrate that distinct C-degrons compete for a common site on Das1, indicating a shared mode of recognition. We identify a positively charged pocket within the Das1 leucine-rich repeat domain as the C-degron binding site. Three basic residues at the base of this pocket are essential for Das1 function, likely mediating electrostatic interactions with the C-terminal carboxyl group of the degron, while additional residues contribute to substrate specificity. Comparative analysis reveals that this pocket and its function are conserved across the Saccharomycetaceae family, supporting a conserved role for Das1 in protein quality control.

KaiC, a clock protein in cyanobacteria, cycles between dephosphorylated and phosphorylated states in a 24-hour period in the presence of KaiA and KaiB. We identified the 322nd residue of KaiC as a third example of period-tuning sites. 322nd-site-directed saturation mutagenesis resulted in a variety of KaiC mutants exhibiting either shortened or lengthened cycles. The tunable range of the periods was from approximately 11 to 78 h without significantly compromising temperature compensation. We conducted biochemical analyses of the 322nd variants and examined their predicted structural models. In contrast to another known period-tuning site, where the period decreases sharply as the side-chain volume increases due to mutations, the cycle lengths correlate only modestly with bulkiness at the 322nd residues. The 322nd residue is located in a C-terminal domain of KaiC and influences ATPase cycles in both the C-terminal domain and an N-terminal domain through its interaction with a flexible loop connecting the two domains. The structural models predict that placing less bulky but polar side chains, such as serine and threonine, at the 322nd position leads to the formation of a hydrogen-bonding network between that site and the loop. This reduces the mobility of the loop, resulting in the longer cycles due to decreases in the ATPase activity of the N-terminal domain. Conversely, placing bulky residues such as phenylalanine at the 322nd position appears to alter the loop structure, shortening the periods by enhancing the ATP activities of both the domains. The third period-tuning mechanism is distinct from other known mechanisms. Significance StatementA Kai-protein clock system serves as a model for studying how long circadian rhythms are achieved. We identified the 322nd residue of KaiC as a third example of period-tuning sites that allow tuning of the period in either long- and short-period directions. The third period-tuning mechanism differs from the two previously known types in several respects. Previous studies have suggested that the ATPase activity in an N-terminal domain of KaiC is the primary regulator of the period. On the other hand, the 322nd residues of KaiC can affect the period by activating the ATPase cycle in its C-terminal domain. Our findings will stimulate future studies on the period-tuning mechanism mediated by the ATPase activity in the C-terminal domain of KaiC.

The radical S-adenosylmethionine (SAM) superfamily comprises more than 800,000 enzymes that use [Fe4S4] clusters to initiate radical chemistry that mediates an exceptionally broad range of chemical transformations. Within this superfamily, cobalamin (Cbl)-dependent radical SAM enzymes constitute a major subclass predominantly associated with methylation reactions. However, several notable members catalyze non-methylase reactions, for which the mechanistic role of Cbl is poorly understood. Bacteriochlorophyll biosynthesis enzyme BchE is a Cbl-dependent radical SAM enzyme that catalyzes a six-electron oxidation of Mg-protoporphyrin IX monomethylester (MPE) to protochlorophyllide (PChlide), installing a ketone and forming the fifth ring of bacteriochlorophyll under anaerobic conditions. Although prior in vivo and in vitro studies have demonstrated a requirement for Cbl, SAM, and a low-potential reductant, detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme. Here, we report the successful isolation and spectroscopic characterization of BchE, enabling the first in vitro reconstitution of its enzymatic activity. Using both chemical and biological reducing systems, we observe the formation of PChlide along with proposed reaction intermediates and several off-pathway products. These results provide new insight into the oxidative chemistry mediated by Cbl in non-methylase radical SAM enzymes and establish BchE as a tractable model for elucidating how cobalamin is deployed in this understudied subclass.

Phycobiliproteins form oligomeric assemblies essential for photosynthetic light harvesting. Here, we engineered phycocyanin (TeCPC) and allophycocyanin (TeAPC) from Thermosynechococcus elongatus to stabilize defined trimers by inhibiting hexamer formation. Structure-guided substitutions at conserved glycine residues (TeCPC G29R, TeAPC G21R) introduce steric hindrance at the hexamer interface. Recombinant expression in Escherichia coli produced holoproteins with native-like chromophorylation. Biophysical and structural analyses confirmed homogeneous trimer formation and absence of higher-order assemblies. Thermal measurements indicated cooperative unfolding, supporting structural uniformity. These engineered trimers provide robust models for studying energy transfer in phycobiliproteins.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Coenzyme A is an essential cofactor synthesized from pantothenate, cysteine, and ATP, and is involved in numerous processes of cellular metabolism through its ability to carry activated acyl groups. Coenzyme A participates in catabolism of carbohydrate, fat and amino acids; biosynthesis of fatty acids, cholesterol and heme; and protein modification including acetylation and 4-phosphopantetheinylation. Despite CoAs critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. One reason for this gap is that many acyl-CoA species are analytically challenging to measure due to factors including instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths. In addition, most current methods do not support analysis of CoA isotopic labeling, which is required to quantify CoA synthesis rate or to measure absolute concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains, then CoA is derivatized on the thiol with N-ethylmaleimide (NEM). Following protein precipitation and solid phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for both pure standards and tissue samples. We applied this method to measure total CoA concentration in five tissues from male and female mice, and total CoA synthesis rate in mouse liver via infusion of 13C-15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and isotopic labeling to enable study of total CoA synthesis rates and concentrations in health and disease.

The de novo design of enzymes critically tests our understanding of natural enzymes and enables design of novel catalysts. Here, we identify the features responsible for the catalytic efficiency of a highly proficient de novo enzyme generated through computational design and optimized by directed evolution. Computational, spectroscopic, and biochemical studies reveal successfully designed features, including precise alignment of catalytic residues, transition state stabilization, and environmental tuning. In the most evolved enzyme, the binding of a transition state analog also led to widespread increases in backbone rigidity and conformational stability throughout the protein, except within a helix near the active site entrance, where the introduction of Gly and Pro increased dynamics and catalytic activity. Thus, the entire protein contributes to catalysis in the most optimized enzyme. These studies provide principles for designing efficient enzymes.

Forster resonance energy transfer (FRET) is a physicochemical phenomenon involving non-radiative energy transfer between donor and acceptor fluorophores. While FRET efficiency primarily depends on the proximity between fluorophores, additional factors also substantially influence the efficiency in living cells. However, how non-distance factors modulate live-cell FRET efficiency remains poorly understood. Here, we report the significant role of N- and C-terminal topology in determining live-cell FRET efficiency, independent of fluorophore proximity, donor variants, and subcellular compartment. Using acceptor photobleaching and sensitized emission measurements in living cells, we found that FRET efficiencies of mCherry-EGFP or mCherry-EYFP (acceptor-donor) were significantly higher than those of EGFP-mCherry or EYFP-mCherry (donor-acceptor), respectively. These efficiencies were comparable between the nucleus and cytoplasm. An orientation index analysis showed that the acceptor-donor configuration is more favorable than the donor-acceptor configuration regardless of donor variants and subcellular localization. FRET efficiencies were also higher with EYFP than with EGFP as the donor. AlphaFold2-based structural modeling suggested similar proximity with structurally heterogeneous and loosely constrained geometry of donor and acceptor fluorophores. Collectively, these results demonstrate that topological arrangement, rather than simple distance considerations, plays a significant role in FRET efficiency in living cells, providing molecular implications for the design of intramolecular FRET-based biosensors.