Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Improvement of substrate specificity of the direct electron transfer type FAD-dependent glucose dehydrogenase catalytic subunit.

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing-including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme's high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its specificity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH's small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475.The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in Vmax towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5mM) of 4.19 U mg-1, as compared to the wild type, 86.6 U mg-1. This resulting increase in specificity lead to an 81.7gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9gal/glc % activity at 5mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose.

The cytochrome P450 subfamilies CYP392A and CYP392D are key players in acaricide metabolism in Tetranychusurticae.

The spider mite Tetranychus urticae is a major agricultural pest with a global distribution, extremely diverse host range and a remarkable ability to develop resistance to a wide variety of acaricides. P450 mono-oxygenases have been frequently associated with resistance development in this species. In particular enzymes of the CYP392A-subfamily were shown to metabolize a number of key acaricides, including abamectin, amitraz, fenpyroximate and the active metabolite of pyflubumide. However, transcriptomic studies comparing highly resistant and susceptible populations have often revealed high expression of members of the CYP392D-subfamily, but these have been only poorly studied. Here, we conducted a meta-analysis of gene expression data of 20 populations and identified two key enzymes of this family, CYP392D2 and CYP392D8, whose expression is associated with resistance. We subsequently functionally expressed these enzymes, together with CYP392A11 and CYP392A16 as known metabolizers, and compared their potential to accept a wide diversity of acaricides as substrate. This study overall confirms previous discovered substrates for CYP392A11 and CYP392A16, but also reveals unreported metabolic activity towards new acaricides. These include carbaryl, chlorpyrifos and etoxazole for CYP392A16 and carbaryl, chlorpyrifos and NNI-0711-NH pyflubumide for CYP392A11. For the newly studied CYP392D-family, we show that CYP392D2 metabolizes pyridaben, fenpyroximate, etoxazole and chlorpyrifos, while CYP392D8 metabolizes carbaryl, fenazaquin and tebufenpyrad. Last, we observed that both CYP392A- and CYP392D-subfamily enzymes activate chlorpyrifos to its corresponding oxon. Our study indicates that there is both overlap and specificity in the activity of A- and D-subfamily enzymes against acaricides and model substrates. With the recent advent of highly efficient CRISPR/Cas9 gene editing protocols in T. urticae, the way is now paved to conduct further genetic experiments revealing and quantifying the role of these enzymes in the resistance phenotype in field populations.

Deep mutational scanning of CYP2C19 in human cells reveals a substrate specificity-abundance tradeoff.

The Cytochrome P450s (CYPs) enzyme family metabolizes ∼80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across CYPs is challenging. Even closely related CYPs like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using Variant Abundance by Massively Parallel sequencing (VAMP-seq), we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for CYP function and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the two homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes.

Substrate Specificity of GH29 α-L-Glucosidases from Cecembia lonarensis.

We recently found two α-L-glucosidases, which can hydrolyze p-nitrophenyl α-L-glucopyranoside (PNP L-Glc) rather than p-nitrophenyl α-L-fucopyranoside, in glycoside hydrolase family 29. This study evaluated their substrate specificity for p-nitrophenyl α-L-rhamnopyranoside (PNP L-Rha), α-L-quinovopyranoside (PNP L-Qui), and α-L-xylopyranoside (PNP L-Xyl), of which structure is similar to PNP L-Glc. The two α-L-glucosidases had little activity toward PNP L-Rha. They exhibited higher k cat/K m values for PNP L-Qui but smaller for PNP L-Xyl than for PNP L-Glc. The molecular docking studies indicated that these specificities were correlated well with the active-site structure of the α-L-glucosidases. The finding that α-L-quinovoside, which has been suggested to occur in nature, is also a substrate for α-L-glucosidases indicates that this enzyme are not solely dedicated to α-L-glucoside hydrolysis.

Co-occurrence between key HAB species and particle-attached bacteria and substrate specificity of attached bacteria in the coastal ecosystem.

The ecological dynamics of particle-attached bacteria (PAB) were observed through changes in the core phytoplankton phycosphere, and were associated with the dynamics of free-living bacteria (FLB) using metabarcoding and microscopic analyses over 210 days (with weekly sampling intervals) in the Jangmok coastal ecosystem, South Korea. Cluster analysis and non-metric multidimensional scaling classified the phytoplankton community into six groups comprising core phytoplankton species, including the harmful algal species Akashiwo sanguinea (dinoflagellate) in late autumn, Teleaulax amphioxeia (cryptomonads) in early winter and spring, Skeletonema marinoi-dohrnii complex (diatom) in winter, Pseudo-nitzschia delicatissima (diatom) in early spring, and diatom complexes such as Chaetoceros curvisetus and Leptocylindrus danicus in late spring. We identified 59 and 32 indicators in PAB and FLB, respectively, which rapidly changed with the succession of the six core phytoplankton species. The characteristics of PAB were mainly divided into "Random encounters" or "Attraction of motivation by chemotaxis." When Akashiwo sanguinea bloomed, bacteria of the genera Kordiimonas and Polaribacter, which are commonly observed in PAB and FLB, indicated "Random encounter" characteristics. In addition, Sedimenticola of PAB was uniquely presented in Akashiwo sanguinea, exhibiting characteristics of "Attraction of motivation by chemotaxis." In contrast, FLB followed the strategy of "Random encounters" because it was not affected by specific habitats and energy sources. Thus, many common bacteria were PAB and FLB, thereby dictating the bacteria's strategy of "Random encounters." "Attraction of motivation by chemotaxis" has characteristics of the species-specific interactions between PAB and specific harmful algal species, and is potentially influenced by organic matter of core phytoplankton cell surface and/or EPS released from phytoplankton.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients.

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90ß, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1-/- cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Multi-method analysis revealed the mechanism of substrate selectivity in NHase: A gatekeeper residue at the activity center.

Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes.

Enhancing catalytic efficiency of Bacillus subtilis laccase BsCotA through active site pocket design.

BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 105 M-1 s-1 to 1.18 × 106 M-1 s-1, without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA's ability to accommodate the 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced kcat. Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases' active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. KEY POINTS: • Active site pocket design that enhanced BsCotA laccase efficiency • 7.7-fold improved in catalytic rate • All tested variants retain thermal stability.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

Probing the nucleobase selectivity of RNA polymerases with dual-coding substrates.

Formycin A (FOR) and Pyrazofurin A (PYR) are nucleoside analogues with antiviral and antitumor properties. They are known to interfere with nucleic acid metabolism, but their direct effect on transcription is less understood. We explored how RNA polymerases (RNAPs) from bacteria, mitochondria, and viruses utilize FOR, PYR, and oxidized purine nucleotides. All tested polymerases incorporated FOR in place of adenine and PYR in place of uridine. FOR also exhibited surprising dual-coding behavior, functioning as a cytosine substitute, particularly for viral RNAP. In contrast, 8-oxoadenine and 8-oxoguanine were incorporated in place of uridine in addition to their canonical Watson-Crick codings. Our data suggest that the interconversion of canonical anti- and alternative syn-conformers underlies dual-coding abilities of FOR and oxidized purines. Structurally distinct RNAPs displayed varying abilities to utilize syn-conformers during transcription. By examining base pairings that led to substrate incorporation and the entire spectrum of geometrically compatible pairings, we have gained new insights into the nucleobase selection processes employed by structurally diverse RNAPs. These insights may pave the way for advancements in antiviral therapies.

Several orphan solute carriers functionally identified as organic cation transporters: Substrates specificity compared with known cation transporters.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations are still unknown. To identify these transporters, genes of 10 known OCTs and 18 orphan solute carriers (SLC) were overexpressed in HEK293 cells and characterized concerning their transport activities with a broad spectrum of low molecular weight substances emphasizing organic cations. Several SLC35 transporters and SLC38A10 significantly enhanced the transport of numerous relatively hydrophobic organic cations. Significant organic cation transport activities have been found in gene families classified as transporters of other substance classes. For instance, SLC35G3 and SLC38A10 significantly accelerated the uptake of several cations, such as clonidine, 3,4-methylenedioxymethamphetamine, and nicotine, which are known as substrates of a thus far genetically unidentified proton/organic cation antiporter. The transporters SLC35G4 and SLC35F5 stood out by their significantly increased choline uptake, and several other SLC transported choline together with a broader spectrum of organic cations. Overall, there are many more polyspecific organic cation transporters than previously estimated. Several transporters had one predominant substrate but accepted some other cationic substrates, and others showed no particular preference for one substrate but transported several organic cations. The role of these transporters in biology and drug therapy remains to be elucidated.

Structural Insights into the Substrate Recognition and Catalytic Mechanism of a GH16 ßκ-Carrageenase from Wenyingzhuangia fucanilytica.

Understanding the substrate specificity of carrageenases has long been of interest in biotechnology applications. So far, the structural basis of the ßκ-carrageenase that hydrolyzes furcellaran, a major hybrid carrageenan, remains unclear. Here, the crystal structure of Cgbk16A_Wf, as a representative of the ßκ-carrageenase from GH16_13, was determined, and the structural characteristics of this subfamily were elucidated for the first time. The substrate binding mode was clarified through a structure analysis of the hexasaccharide-bound complex and molecular docking. The binding pocket involves a conserved catalytic motif and several specific residues associated with substrate recognition. Functions of residues R88, E290, and E184 were validated through site-directed mutagenesis. Comparing ßκ-carrageenase with κ-carrageenase, we proposed that their different substrate specificities are partly due to the distinct conformations of subsite -1. This research offers a comprehensive understanding of the recognition mechanism of carrageenases and provides valuable theoretical support for enzyme modification and carrageenan oligosaccharide preparation.

Docking interactions determine substrate specificity of members of a widespread family of protein phosphatases.

How protein phosphatases achieve specificity for their substrates is a major outstanding question. PPM family serine/threonine phosphatases are widespread in bacteria and eukaryotes, where they dephosphorylate target proteins with a high degree of specificity. In bacteria, PPM phosphatases control diverse transcriptional responses by dephosphorylating anti-anti-sigma factors of the STAS domain family, exemplified by Bacillus subtilis phosphatases SpoIIE, which controls cell-fate during endospore formation, and RsbU, which initiates the general stress response. Using a combination of forward genetics, biochemical reconstitution, and AlphaFold2 structure prediction, we identified a conserved, tripartite substrate docking interface comprised of three variable loops on the surface of the PPM phosphatase domains of SpoIIE and RsbU that recognize the three-dimensional structure of the substrate protein. Nonconserved amino acids in these loops facilitate the accommodation of the cognate substrate and prevent dephosphorylation of the noncognate substrate. Together, single-amino acid substitutions in these three elements cause an over 500-fold change in specificity. Our data additionally suggest that substrate-docking interactions regulate phosphatase specificity through a conserved allosteric switch element that controls the catalytic efficiency of the phosphatase by positioning the metal cofactor and substrate. We hypothesize that this is a generalizable mechanistic model for PPM family phosphatase substrate specificity. Importantly, the substrate docking interface with the phosphatase is only partially overlapping with the much more extensive interface with the upstream kinase, suggesting the possibility that kinase and phosphatase specificity evolved independently.

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

The structure of DNA methyltransferase DNMT3C reveals an activity-tuning mechanism for DNA methylation.

DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologs DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A, and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A, and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, explaining the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unraveling a regulatory mechanism by which evolutionary conservation and diversification fine-tune the activity of de novo DNA methyltransferases.

Functional Diversity and Engineering of the Adenylation Domains in Nonribosomal Peptide Synthetases.

Nonribosomal peptides (NRPs) are biosynthesized by nonribosomal peptide synthetases (NRPSs) and are widely distributed in both terrestrial and marine organisms. Many NRPs and their analogs are biologically active and serve as therapeutic agents. The adenylation (A) domain is a key catalytic domain that primarily controls the sequence of a product during the assembling of NRPs and thus plays a predominant role in the structural diversity of NRPs. Engineering of the A domain to alter substrate specificity is a potential strategy for obtaining novel NRPs for pharmaceutical studies. On the basis of introducing the catalytic mechanism and multiple functions of the A domains, this article systematically describes several representative NRPS engineering strategies targeting the A domain, including mutagenesis of substrate-specificity codes, substitution of condensation-adenylation bidomains, the entire A domain or its subdomains, domain insertion, and whole-module rearrangements.

Substrate specificity modification of paraben hydrolase and tannase from Aspergillus oryzae.

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.

Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Oligomeric Structure of Yeast and Other Invertases Governs Specificity.

Invertases, or ß-fructofuranosidases, are metabolic enzymes widely distributed among plants and microorganisms that hydrolyze sucrose and release fructose from various substrates. Invertase was one of the earliest discovered enzymes, first investigated in the mid-nineteenth century, becoming a classical model used in the primary biochemical studies on protein synthesis, activity, and the secretion of glycoproteins. However, it was not until 20 years ago that a member of this family of enzymes was structurally characterized, showing a bimodular arrangement with a ß-propeller catalytic domain, and a ß-sandwich domain with unknown function. Since then, many studies on related plant and fungal enzymes have revealed them as basically monomeric. By contrast, all yeast enzymes in this family that have been characterized so far have shown sophisticated oligomeric structures mediated by the non-catalytic domain, which is also involved in substrate binding, and how this assembly determines the particular specificity of each enzyme. In this chapter, we will review the available structures of yeast invertases to elucidate the mechanism regulating oligomer formation and compare them with other reported dimeric invertases in which the oligomeric assembly has no apparent functional implications. In addition, recent work on a new family of invertases with absolute specificity for the α-(1,2)-bond of sucrose found in cyanobacteria and plant invertases is highlighted.