Wild-type Enzyme Research Articles

Abstract PO2-18-04: STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents

Abstract Activating mutations in PI3Kα are among the most prevalent genetic aberrations in breast cancer. Constitutive signaling of PI3Kα is an established oncogenic driver in these cancers, as these mutations are predictors of response to alpelisib, an approved PI3Kα inhibitor, which inhibits both wild type and mutant PI3Kα. While inhibiting PI3Kα has proven to be efficacious, the concomitant inhibition of wild type enzyme in normal tissue results in metabolic dysfunction and ultimately dose-limiting toxicities. Selectively targeting the mutant PI3Kα enzyme is expected to inhibit tumor growth while sparing normal tissues, thus increasing the therapeutic index compared to non-mutant selective PI3Kα inhibitors. Therefore, we have developed STX-478, an allosteric, CNS penetrant, mutant-selective PI3Kα inhibitor. In a high-throughput viability screen, STX-478 selectively inhibited growth of cell lines with kinase domain and helical domain mutations in PI3Kα. In a panel of PI3Kα mutant CDX and PDX models, STX-478 provided efficacy that was similar or superior to higher than clinically achievable doses of alpelisib. Robust suppression of pharmacodynamic (PD) markers, pAKT, was observed in tumors, but not in skeletal muscle. Importantly, the efficacy achieved with STX-478 occurred at doses that did not cause metabolic dysfunction, in contrast to alpelisib which caused insulin release and suppressed glucose uptake in skeletal muscle in a 13C glucose tolerance test. Collectively, these data indicate that STX-478 has an expanded therapeutic window to selectively target mutant PI3Kα in vivo. In efficacy studies using human tumor xenograft models, STX-478 caused tumor regressions as a single agent. In multiple advanced ER+ breast cancer PDX models including models that are insensitive to the approved CDK4/6 inhibitor palbociclib, STX-478 combined with standards of care was highly efficacious and well tolerated. In a representative PDX model, STX-478 combined with the ER degrader fulvestrant and palbociclib was well tolerated for > 90 days of dosing in mice and resulted in durable tumor regressions that were superior to STX-478 alone. In conclusion, these data demonstrate STX-478 is a highly efficacious, well tolerated, mutant-specific PI3Kα inhibitor that can be combined safely with standards of care in breast cancer and provide the therapeutic benefits of PI3Kα inhibition without the side effects associated with inhibiting WT PI3Kα. These properties combined with the potential for CNS penetration and excellent pharmacokinetic profile in higher species make STX-478 a potentially best-in-class mutant-selective PI3Kα inhibitor. STX-478 is currently being evaluated in a Phase I clinical trial (NCT05768139). Citation Format: Trang Tieu, Leonard Buckbinder, David St. Jean, Samantha Manimala, Gregory Dowdell, Michael Huff, Jacob Alltucker, Erica Jackson, Angel Guzman-Perez, Darrin Stuart. STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-04.

Abstract PO3-25-09: OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models

Abstract Mutations in PI3Kα lead to constitutive activation of the PI3K/AKT/mTOR pathway and are highly prevalent in cancer. PI3KαH1047R is the most common amino acid substitution mutation and represents approximately one-third of all PI3Ka mutations, with 15% of breast cancers having a PI3KαH1047R mutation. In breast cancer, PI3KαH1047R mutations are associated with resistance to targeted therapies, including HER2- and estrogen receptor (ER)- targeted agents. Although targeting PI3Kα in cancer is a therapeutically proven strategy, with the currently approved drug alpelisib showing clinical efficacy alone or in combination with other therapies, treatment with non-mutant selective PI3Kα inhibitors such as alpelisib is associated with significant toxicities such as hyperglycemia, due to on-target inhibition of the wild-type enzyme. This often leads to dose modification or discontinuation. Therefore, there is a significant need to develop new PI3Kα-targeted therapies that can avoid or minimize on-target toxicity and improve the safety and clinical benefit for patients. OKI-219 is an orally bioavailable, PI3KαH1047R mutant-selective inhibitor, with ~100-fold cellular selectivity for the H1047R mutation over wild-type. OKI-219 demonstrates selectivity across a panel of 412 kinases, with no other kinases showing >30% inhibition at 1 µM. In vitro, OKI-219 drives decreased phosphorylated AKT (pAKT) and decreased proliferation specifically in PI3KαH1047R mutant cell lines across several tumor types, including breast cancer. In vivo, as a monotherapy, OKI-219 demonstrates dose dependent antitumor activity in multiple human tumor xenograft models, including the H1047R mutant breast cancer CDX models T47D and HCC1954 and several PDX models, with regression rates up to 100% at doses that show no sign of the metabolic dysfunction (increased glucose or insulin) associated with wild-type protein inhibition. The in vivo antitumor activity correlates with pathway inhibition, measured by decreased pAKT in tumors. Moreover, OKI-219 dosed in combination with the selective estrogen receptor degraders (SERDs) fulvestrant, elacestrant or camizestrant showed significant combination benefit leading to tumor regressions of up to 100% in the ER+ H1047R mutant breast cancer model xxT47D, at doses where no regressions were observed with single agent treatment. Dosing OKI-219 in combination with HER2-inhibitors, such as tucatinib, led to tumor regressions in the ER-HER2+ and H1047R mutant breast cancer CDX model HCC1954, also at doses where no regressions were observed with single agent treatment. These data indicate that OKI-219 offers improved efficacy and a wider therapeutic window compared to non-mutant selective PI3Kα inhibitors. OKI-219 is advancing into clinical trials. Citation Format: Molly A. Taylor, Qian Zhao, David Mareska, Maria Hoh, Yevgeniy Izrayelit, Kevin Litwiler, Mark L Boys, Rich Woessner, Duncan Walker, James D Winkler, Jennifer Diamond. OKI-219, a PI3KαH1047R-mutant-selective inhibitor demonstrates efficacy as a single agent and drives combination responses with standard of care therapies in pre-clinical PI3KαH1047R mutant breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-09.

Improved Enzymatic Production of the Fucosylated Human Milk Oligosaccharide LNFP II with GH29B α-1,3/4-l-Fucosidases.

Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Semirational Design Strategy To Enhance the Thermostability and Catalytic Activity of Cytochrome P450 105D7 for the Degradation of the Pharmaceutically Active Compounds: Diclofenac.

Pharmaceutically active compounds are an important category of emerging pollutants, and their biological transformation processes in the environment are crucial for understanding and evaluating the migration, transformation, and environmental fate of emerging pollutants. The cytochrome P450 105 enzyme family has been proven to play an important role in the degradation of exogenous environmental pollutants. However, its thermostability and catalytic activity still need to be improved to better adapt to complex environmental conditions. This work elucidates the key mechanisms and important residues of the degradation reaction through multiple computational strategies, establishes a mutation library, and obtains 21 single-point mutation designs. Experimental verification showed that 16 single mutants had enhanced thermostability, with the R89F and L197Y mutants showing the highest increases in thermostability at 135 and 119% relative to the wild-type enzyme, respectively. Additionally, as a result of the higher specific activity of D390Q, it was selected for combination mutagenesis, ultimately resulting in three combination mutants (R89F/L197Y, R89F/D390Q, and R89F/L197Y/D390Q) with enhanced thermostability and catalytic activity. This study provides a modification approach for constructing efficient enzyme variants through semirational design and can contribute to the development of control technologies for emerging pollutants.

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity.

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver of the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being >1000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, and some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops and changes to their conformations, as revealed by a suite of crystal structures. This suggests that a specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining the enzyme activity profile.

Biochemical and structural impact of two novel missense mutations in cystathionine β-synthase gene associated with homocystinuria.

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Hydrophobic substrate binding pocket remodeling of echinocandin B deacylase based on multi-dimensional rational design

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates.

Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-β-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-β-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-β-d-glucanase, enabling the hydrolysis of (1,3;1,4)-β-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-β-d-glucan endohydrolase [(1,3;1,4)-β-d-glucanase] isoenzyme EII (HvEII) and (1,3)-β-d-glucan endohydrolase [(1,3)-β-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-β-d-glucanase and one variant that hydrolyzed both (1,3)-β-d-glucans and (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-β-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-β-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-β-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-β-d-glucans or (1,3)-β-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-β-d-glucans and (1,3)-β-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Biochemical, structural, and computational analyses of two new clinically identified missense mutations of ALDH7A1

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes a step of lysine catabolism. Certain missense mutations in the ALDH7A1 gene cause pyridoxine dependent epilepsy (PDE), a rare autosomal neurometabolic disorder with recessive inheritance that affects almost 1:65,000 live births and is classically characterized by recurrent seizures from the neonatal period. We report a biochemical, structural, and computational study of two novel ALDH7A1 missense mutations that were identified in a child with rare recurrent seizures from the third month of life. The mutations affect two residues in the oligomer interfaces of ALDH7A1, Arg134 and Arg441 (Arg162 and Arg469 in the HGVS nomenclature). The corresponding enzyme variants R134S and R441C (p.Arg162Ser and p.Arg469Cys in the HGVS nomenclature) were expressed in Escherichia coli and purified. R134S and R441C have 10,000- and 50-fold lower catalytic efficiency than wild-type ALDH7A1, respectively. Sedimentation velocity analytical ultracentrifugation shows that R134S is defective in tetramerization, remaining locked in a dimeric state even in the presence of the tetramer-inducing coenzyme NAD+. Because the tetramer is the active form of ALDH7A1, the defect in oligomerization explains the very low catalytic activity of R134S. In contrast, R441C exhibits wild-type oligomerization behavior, and the 2.0 Å resolution crystal structure of R441C complexed with NAD+ revealed no obvious structural perturbations when compared to the wild-type enzyme structure. Molecular dynamics simulations suggest that the mutation of Arg441 to Cys may increase intersubunit ion pairs and alter the dynamics of the active site gate. Our biochemical, structural, and computational data on two novel clinical variants of ALDH7A1 add to the complexity of the molecular determinants underlying pyridoxine dependent epilepsy.

Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Effect of (R)-2-Amino-6-(1R,2S)-1,2-Dihydroxypropyl)-5,6,7,8-Tetrahydropterin-4(3H)-One and Its Structural Analogues on the Temperature Stability of Tryptophan Hydroxylase 2 with the P447R Mutation.

The enzyme tryptophan hydroxylase 2 (TPH2) catalyzes the hydroxylation of L-tryptophan to L-5-hydroxytryptophan (5-HTP), the first and the key step in 5-HT synthesis in the mammalian brain. Mutations in the human Tph2 gene reducing enzyme activity increase the risk of psychopathology. Pharmacological chaperones are small molecules that can specifically bind to mutant protein molecules, restore their disturbed 3D structure to the native state, and increase their stability and functional activity. The chaperone activity of (R)-2-amino-6-(1R,2S)-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one (BH4) is expressed by increasing the in vitro thermal stability of mutant tyrosine hydroxylase and phenylalanine hydroxylase molecules which are similar to TPH2 in their structure and characteristics. The P447R substitution in the mouse TPH2 molecule results in a 2-fold decrease in enzyme activity in their brains. We studied the effect of this mutation on the TPH2 thermal stability, as well as on the ability of BH4 and its 8 structural analogues to increase the thermal stability of the mutant TPH2 from midbrain extracts of BALB/C mice. Temperature stability was studied by the decrease in enzyme activity during its heating for 2 min at increasing temperatures and was evaluated by the T50 value that is the temperature at which the enzyme activity decreased by half. For the mutant TPH2, the T50 value was decreased compared to the wild type enzyme. BH4 and its closest structural analogue, 6-methyl-5,6,7,8-tetrahydropterin, increased the T50 value, i.e., exhibited chaperone activity. Other close BH4 analogs, 6,7-dimethyl-5,6,7,8-tetrahydropterin and folic acid, were not effective. It can be assumed that BH4 can be effective in the treatment of mental disorders caused by mutations in the Tph2 gene.

Heterozygous gain of function variant in GUCY1A2 may cause autonomous ovarian hyperfunction.

The purpose of this study was to characterize the phenotype associated with a de novo gain-of-function variant in the GUCY1A2 gene. An individual carrying the de novo heterozygous variant c.1458G>T p.(E486D) in GUCY1A2 was identified by exome sequencing. The effect of the corresponding enzyme variant α2E486D/β1 was evaluated using concentration-response measurements with wild-type enzyme and the variant in cytosolic fractions of HEK293 cells, UV-vis absorbance spectra of the corresponding purified enzymes, and examination of overexpressed fluorescent protein-tagged constructs by confocal laser scanning microscopy. The patient presented with precocious peripheral puberty resembling the autonomous ovarian puberty seen in McCune-Albright syndrome. Additionally, the patient displayed severe intellectual disability. In vitro activity assays revealed an increased nitric oxide affinity for the mutant enzyme. The response to carbon monoxide was unchanged, while thermostability was decreased compared to wild type. Heme content, susceptibility to oxidation, and subcellular localization upon overexpression were unchanged. Our data define a syndromic autonomous ovarian puberty likely due to the activating allele p.(E486D) in GUCY1A2 leading to an increase in cGMP. The overlap with the ovarian symptoms of McCune-Albright syndrome suggests an impact of this cGMP increase on the cAMP pathway in the ovary. Additional cases will be needed to ensure a causal link.

Cooperative nucleic acid binding by Poly ADP-ribose polymerase 1

Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1’s normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T20 and U20 oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T20 and U20 oligonucleotides show that PARP’s binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1’s ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.

The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases

We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use β-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 Å from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1–4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.

Modulating the Acceptor Preference of His‐C‐Geranyltransferase LimF

AbstractLipidation stands as a pivotal strategy for enhancing the metabolic stability of target peptides. Prenyltransferases in cyanobactin biosynthesis have garnered significant attention as potential peptide lipidation biocatalysts because of their exceptional regio‐ and chemoselectivity. However, these enzymes often exhibit a biased preference for certain acceptor substrates, requiring specific amino acids adjacent to the modifying residue. In this study, we demonstrate the structure‐guided engineering of LimF, a His‐C‐geranyltransferase, to broaden its peptide substrate tolerance. By altering key residues in the peptide‐binding pocket, we created a LimF variant capable of modifying sequence motifs previously inaccessible to the wildtype enzyme. The variant successfully modified some previously unfavored sequence motifs in artificial peptide substrates and bioactive peptide agents, validating the engineered substrate scope. With the discovery of novel peptide prenyltransferases, this approach would lead to a more comprehensive toolbox of peptide prenylation biocatalysts.

Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis.

Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.

Adapting an acyl CoA ligase from Metallosphaera sedula for lactam formation by structure-guided protein engineering

The CoA ligase from Metallosphaera sedula (MsACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of MsACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of MsACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction.

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L−1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.