Stability of tryptophan-containing LOs in flaxseed oil and their response towards γ-tocopherol.

Linusorbs (LOs), significantly influence oil quality and sensory properties of flaxseed oil. Trp-containing LOs exhibit distinct oxidative behavior when γ-tocopherol (γ-T) is present. Polar fractions of crude flaxseed oil were stripped via silica absorption, and reintroduced (LO and γ-T) separately into the oil matrix to investigate their interaction during storage. Compared with crude oil, LOs account for 18.49% reduction of p-anisidine value, while LOs with γ-T contributed to most of the endogenous antioxidant effect in crude oil. γ-T was found to suppress oxidation of Trp-containing LO at early stage (Met form), while facilitate oxidation while at their mid-stage (MetO form, Methionine sulfoxide). In vitro oxidation shows that CLD more likely cleaved into peptide fragments, while few products retain intact ring structures. LC-MS/MS analysis and silicon simulation revealed proximity between MetO and Trp residues, facilitating inter- or intra-molecular reactions and ring structure rupture. Remarkably, the presence of γ-T facilitate these phenomena.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Ultraviolet Resonance Raman Spectra of Serum Albumins.

The ultraviolet resonance Raman (UVRR) spectra of the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) in an aqueous solution are compared with the aim to distinguish between them based on their very similar amino acid composition and structure and to obtain signals from tryptophan that has only very few residues. Comparison of the protein spectra with solutions of tryptophan, tyrosine, and phenylalanine in comparative ratios as in the two proteins shows that at an excitation wavelength of 220 nm, the spectra are dominated by the strong resonant contribution from these three amino acids. While the strong enhancement of two and one single tryptophan residue in BSA and HSA, respectively, results in pronounced bands assigned to fundamental vibrations of tryptophan, its weaker overtones and combination bands do not play a major role in the spectral range above 1800 cm-1. There, the protein spectra clearly reveal the signals of overtones and combination bands of phenylalanine and tyrosine. Assignments of spectral features in the range of Raman shifts from 3800 to 5100 cm-1 to combinations comprising fundamentals and overtones of tyrosine were supported by spectra of amino acid mixtures that contain deuterated tyrosine. The information in the high-frequency region of the UVRR spectra could provide information that is complementary to near-infrared absorption spectroscopy of the proteins.

Safety concerns regarding impurities in L-Tryptophan associated with eosinophilia myalgia syndrome.

L-tryptophan is one of the essential amino acids in humans and across the animal kingdom. It has been widely used as a feed additive for domestic animals and is also administered through dietary supplements in humans. Safety concerns have been raised however since a disease known as eosinophilia-myalgia syndrome (EMS) was reported to be related to L-tryptophan supplements. EMS is a rare condition characterized by inflammation in various organ systems including the muscles, skin, and lungs. Through several studies, it has been speculated that the six components generated during the process of L-tryptophan synthesis are related to the induction of EMS. In this review, we discuss the history of EMS and its controversial correlation with L-tryptophan use reported in several studies. Many in vitro and in vivo studies have been conducted to assess the putative correlation between impurities in L-tryptophan preparations and EMS, but no clear and convincing conclusions have been drawn so far.

Role of Charged Amino Acids in Sullying the Fluorescence of Tryptophan or Conjugated Dansyl Probe in Monomeric Proteins.

When Trp/dansyl probe conjugated to a monomeric protein is photoexcited, it is assumed that all emitted fluorescence originates solely from them. In this work, we show that hidden unconventional intrinsic chromophores (called ProCharTS) that originate from confined charge clusters in the protein can contaminate Trp/dansyl emission. Previous work has shown that charge recombination among charge-separated excited states of monomeric proteins, rich in charged residues, can emit weak luminescence (300-700 nm) overlapping with ProCharTS absorption (250-800 nm) and Trp (300-400 nm) and dansyl (400-600 nm) emission. We examine how this overlap taints the fluorescence arising from Trp/dansyl. We compared the effect of dense aqueous solutions of amino acids, Lys/Glu/Asp/Arg/His, on the fluorescence intensity decay/spectrum of N-acetyl-l-tryptophan amide (NATA). Significant broadening on the red side of Trp emission spectrum was observed solely in the presence of lysine, which appeared to be the most potent in altering the mono-exponential fluorescence decay of NATA. Interestingly, NATA in the presence of proteins α3C and dehydrin (DHN1), which are rich in Lys residues, showed substantial deviation from mono-exponential fluorescence decay in contrast to PEST wt and Symfoil-4P pv2, which lack Lys residues. Remarkably, Trp emission spectra among charge-rich proteins like α3W, PEST M1, and DHN1 CW1 were altered on the red side of Trp emission. Emission spectrum of dansyl-labeled human serum albumin (HuSA) was broadened and its fluorescence quenched with gradual addition of excess unlabeled HuSA, which displays bountiful ProCharTS luminescence. Our results unveil the additive influence of ProCharTS luminescence on Trp/dansyl emission with no measurable evidence of energy transfer.

Copper(II)-mediated C-H sulphenylation or selenylation of tryptophan enabling macrocyclization of peptides.

Cu(II)-mediated C-H sulphenylation or selenylation of Trp indole by a derivative of cysteine or selenocysteine enables access to the tryptathionine unit or its selenium congener. The mechanism of these protocols, which allow macrocyclization of Trp-containing peptides, has been studied.

Potential Anti-Alzheimer Agents from Guanidinyl Tryptophan Derivatives with Activities of Membrane Adhesion and Conformational Transition Inhibitions.

Guanidinyl tryptophan derivatives TGN1, TGN2, TGN3, and TGN4 were synthesized, and these compounds were shown to possess in vitro inhibitory activity for amyloid aggregation in a previous study. Nevertheless, the influence of the TGN series of compounds on the binding and permeation behaviors of an Aß monomer to the cell membranes was not elucidated. In this study, we investigated the effect of compounds in the TGN series on the behavior of an Aß monomer regarding its toxicity toward the bilayer lipid membrane using molecular dynamics (MD) simulation. MD simulations suggest that TGN4 is a potential agent that can interfere with the movement of the Aß monomer into the membrane. The MM-GBSA result demonstrated that TGN4 exhibits the highest affinity to the Aß1-42 monomer but has the lowest affinity to the bilayer. Moreover, TGN4 also contributes to a decrease in the binding affinity between the Aß1-42 monomer and the POPC membrane. Regarding the results of the binding mode and conformational analyses, a high number of amino-acid residues were shown to provide the binding interactions between TGN4 and the Aß1-42 monomer. TGN4 also reduces the conformational transition of the Aß1-42 monomer by means of interacting with the monomer. The present study presents molecular-level insights into how the TGN series of compounds affect the membrane adsorption and the conformational transition of the Aß1-42 monomer, which could be valuable for the further development of new anti-Alzheimer agents.

Photostability of l-tryptophan in aqueous solution: Effect of atmosphere and antioxidants addition.

l-Tryptophan (l-Trp) is an amino acid important in nutrition, and mainly provided by food supplements. However, it is known to be unstable under light irradiation, which is an issue for the nutrition and feed industry. In the present study, the photostability of l-Trp was studied in acidic aqueous solutions under air and under an inert atmosphere, N2. The photodegradation was followed using UV-visible and fluorescence spectroscopy after photolysis. Moreover, molecular orbitals and bond dissociation energies calculations, and electron spin resonance spectroscopy were performed. From all these results, a photodegradation occurring through a free radical pathway was suggested. Interestingly, several antioxidants were tested to improve the photostability of l-Trp, especially during irradiation under air, since the l-Trp was evidenced to be much less stable under air than under N2. The results showed that sodium benzoate or EDTA were not efficient, but antioxidants such as chlorogenic acid, ascorbic acid or potassium sorbate improved significantly the photostability of l-Trp in acidic solutions.

Dietary supplementation of acetate-conjugated tryptophan alters feed intake, milk yield and composition, blood profile, physiological variables, and heat shock protein gene expression in heat-stressed dairy cows.

The purpose of this study was to investigate the effects of dietary supplementation of rumen-protected tryptophan (RPT) at four levels on milk yield, milk composition, blood profile, physiological variables, and heat shock protein gene expression in dairy cows under conditions of moderate-severe heat stress (MSHS, THI = 80~89). Sixteen early-lactating dairy cows (body weight = 719 ± 66.4 kg, days in milk = 74.3 ± 7.1, milk yield = 33.55 ± 3.74 kg, means ± SEM) were randomly assigned in a factorial arrangement to one of the four treatments: control group (n = 4, no RPT supplementation), 15 g/d RPT (n = 4), 30 g/d RPT (n = 4), or 60 g/d RPT group per cow (n = 4) supplemented to the TMR. A higher dry matter intake (DMI) and milk yield were found in the 30 g RPT group compared with the other groups, and the 3.5% fat-corrected milk yield, energy-corrected milk yield, milk fat, protein, ß-casein, mono-unsaturated fatty acid, and poly-unsaturated fatty acid contents, and serum glucose content were observed in the 30 g RPT group (p < 0.05). The milk lactose concentration was significantly higher in the 30 g RPT group compared with the control and 60 g RPT groups (p < 0.05). The plasma cortisol level was lower, while the serotonin and melatonin concentrations were higher in the 30 g group compared with the other groups (p < 0.05). Heat shock protein (HSP) 70 expression was downregulated in the control and 15 g RPT groups, whereas the expression of HSP90 and HSPB1 remained unchanged among the groups. In particular, the 30 g RPT group was considered to have an improved DMI, milk yield, and lactose concentration, as well as anti-heat stress effects due to the simulation of serotonin and melatonin during MSHS.

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan.

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in the blood. The general consensus is that extracellular Ca2+ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca2+ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca2+ and L-Trp binding sites and conformational changes of the ECD upon Ca2+/L-Trp binding. However, it remains to be understood at the structural level how Ca2+/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca2+- or L-Trp-bound states, and Ca2+/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca2+ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca2+ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca2+. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Tryptophan derived natural marine alkaloids and synthetic derivatives as promising antimicrobial agents.

Antimicrobial resistance has become a major threat to public health worldwide, as pathogenic microorganisms are finding ways to evade all known antimicrobials. Therefore, the demand for new and effective antimicrobial agents is also increasing. Natural products have always played an important role in drug discovery, either by themselves or as inspiration for synthetic compounds. The marine environment is a rich source of bioactive metabolites, and among them, tryptophan-derived alkaloids stand out for their abundance and by displaying a variety of biological activities, with antimicrobial properties being among the most significant. This review aims to reveal the potential of marine alkaloids derived from tryptophan as antimicrobial agents. Relevant examples of these compounds and their synthetic analogues reported in the last decades are presented and discussed in detail, with their mechanism of action and synthetic approaches whenever relevant. Several tryptophan-derived marine alkaloids have shown potent and promising antimicrobial activities, whether against bacteria, fungi, or virus. Synthetic approaches to many of the compounds have been developed and recent methodologies are proving to be efficient. Even though most of the studies regarding the antimicrobial activity are still preliminary, this class of compounds has proven to be worth of further investigation and may provide useful lead compounds for the development of antimicrobial agents. Overall, marine alkaloids derived from tryptophan are revealed as a valuable class of antimicrobials and molecular modifications in order to reduce the toxicity of these compounds and additional studies regarding their mechanism of action are interesting topics to explore in the future.

Flavonoids with inhibitory activity against SARS-CoV-2 3CLpro.

Coronavirus disease 2019 (COVID-19) has been a pandemic disease of which the termination is not yet predictable. Currently, researches to develop vaccines and treatments is going on globally to cope with this disastrous disease. Main protease (3CLpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the good targets to find antiviral agents before vaccines are available. Some flavonoids are known to inhibit 3CLpro from SARS-CoV which causes SARS. Since their sequence identity is 96%, a similar approach was performed with a flavonoid library. Baicalin, herbacetin, and pectolinarin have been discovered to block the proteolytic activity of SARS-CoV-2 3CLpro. An in silico docking study showed that the binding modes of herbacetin and pectolinarin are similar to those obtained from the catalytic domain of SARS-CoV 3CLpro. However, their binding affinities are different due to the usage of whole SARS-CoV-2 3CLpro in this study. Baicalin showed an effective inhibitory activity against SARS-CoV-2 3CLpro and its docking mode is different from those of herbacetin and pectolinarin. This study suggests important scaffolds to design 3CLpro inhibitors to develop antiviral agents or health-foods and dietary supplements to cope with SARS-CoV-2.

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.

Nafion-stabilized black phosphorus nanosheets-maltosyl-ß-cyclodextrin as a chiral sensor for tryptophan enantiomers.

In this work, we have fabricated nafion (NF) stabilized black phosphorus nanosheets (BPNSs) and 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) composite (BPNSs-G2-ß-CD) as novel electrochemical sensoring platform for chiral recognition of tryptophan (Trp) enantiomers. BPNSs-G2-ß-CD composite modified glassy carbon electrode (BPNSs-G2-ß-CD/GCE) was further coated with NF which served as a protective film to immobilize BPNSs-G2-ß-CD on the electrode surface to achieve high stability. Under the optimum conditions, the oxidation peak current ratio of L-Trp to D-Trp (IL/ID) and the difference between the peak potential (ΔEp = ED - EL) were observed to be 1.49 and 20 mV at NF/BPNSs-G2-ß-CD/GCE by square wave voltammetry (SWV). In addition, a linear calibration curve could be obtained for peak current versus Trp enantiomers in the concentration range 0.01-1.00 mM with detection limits of 1.07 µM and 1.71 µM for L-Trp and D-Trp (signal-to-noise ratio of 3, S/N = 3), respectively. The chiral recognition mechanism was also proposed, and the intermolecular hydrogen bonding interactions as well as the hydrophobic-cavity-triggered embedding effect dominated the effective chiral recognition. Moreover, the proposed NF/BPNSs-G2-ß-CD/GCE showed excellent stability, good reproducibility and anti-interference capability. Therefore, the designed chiral sensor is expected to be practically applied for the sensitive recognition of Trp enantiomers in real samples.

Determination of iron(II) and iron(III) via static quenching of the fluorescence of tryptophan-protected copper nanoclusters.

"Tryptophan-coated blue fluorescent copper nanocluster (CuNC@Trp) was prepared by a strategy where Trp acts as both the reducing and capping agent. The fluorescence of the CuNC, with excitation/emission peaks at 340/405 nm, is selectively quenched by iron(II) and iron(III) ions. Studying the mechanism of this interaction revealed that Fe2+ and Fe3+ ions can make a ground state complex with the protecting ligand which can result in quenching of the cluster emission. Structural and optical properties of the modified CuNC were investigated by ESI-MS, DLS, TEM, UV-vis and photoluminescence. The effects of pH value and temperature, time of interaction, and cluster volume were optimized. Under optimized conditions, the probe response is linear in concentration range of 10-1000 µM for Fe(II) and Fe(III) with the relative standard deviations of 0.13 and 0.14% (n = 5) respectively. The respective limits of detection are 3.0 and 2.2 µM. The method was successfully used for determination of trace amount of both ions in spiked water, blood and iron supplement tablets. The results were in good agreement with those obtained by the ICP-AES method." Graphical abstractThe scheme represents the synthesis of CuNC@Trp at basic conditions and at elevated temperature. The emission of the cluster decreases due to static quenching of fluorescence by iron ions.

Antidepressant-Like Effect of ß-Lactolin, a Glycine-Threonine-Tryptophan-Tyrosine Peptide.

The number of patients with mental illnesses, including depression, is rapidly increasing, and daily lifestyle is closely associated with the development of symptoms. Consequently, corrective measures, such as diet-based treatment for diseases, are receiving great attention. We previously showed that ß-lactolin, a ß-lactopeptide of glycine-threonine-tryptophan-tyrosine peptide, inhibits monoamine oxidase and improves memory impairment in mice, but the effects on depression have not been investigated. Here we showed that ß-lactolin improved depression-like behavior via dopamine-D1-like receptor. Orally administered ß-lactolin reduced immobility time in tail suspension test (TST). Pretreatment with SCH23390, dopamine D1-like receptor antagonist, attenuated the reduction in TST by ß-lactolin. These effects were observed by the treatment with whey digest rich in ß-lactolin. In addition, ß-lactolin increased the levels of dopamine in the frontal cortex associated with the depression-like behavior. The present study suggests that supplements or nutraceutical compounds in whey digests (such as ß-lactolin) show antidepressant-like effect.

Water-Mediated Selenium Hydrogen-Bonding in Proteins: PDB Analysis and Gas-Phase Spectroscopy of Model Complexes.

High-resolution X-ray crystallography and two-dimensional NMR studies demonstrate that water-mediated conventional hydrogen-bonding interactions (N-H···N, O-H···N, etc.) bridging two or more amino acid residues contribute to the stability of proteins and protein-ligand complexes. In this work, we have investigated single water-mediated selenium hydrogen-bonding interactions (unconventional hydrogen-bonding) between amino acid residues in proteins through extensive protein data bank (PDB) analysis coupled with gas-phase spectroscopy and quantum chemical calculation of a model complex consisting of indole, dimethyl selenide, and water. Here, indole and dimethyl selenide represent the amino acid residues tryptophan and selenomethionine, respectively. The current investigation demonstrates that the most stable structure of the model complex observed in the IR spectroscopy mimics single water-mediated selenium hydrogen-bonded structural motifs present in the crystal structures of proteins. The present work establishes that water-mediated Se hydrogen-bonding interactions are ubiquitous in proteins and the number of these interactions observed in the PDB is more than that of direct Se hydrogen-bonds present there.

Preventive Effects of Tryptophan-Methionine Dipeptide on Neural Inflammation and Alzheimer's Pathology.

Preventive approaches for age-related memory decline and dementia have become a high priority in the aging society because of the lack of therapeutic approaches. Recent epidemiological studies have reported that fermented dairy products can help prevent dementia. Previously, we identified tryptophan-tyrosine (WY) and tryptophan-methionine (WM) peptides as the suppressants of activation of the primary microglia and showed that WY peptide consumption suppresses inflammation in the brains of Alzheimer's disease model mice. However, the effects of the WM peptide on inflammation in the brain and Alzheimer's pathology have not been investigated. Here, we evaluated the effect of WM peptide consumption on Alzheimer's disease model (5×FAD) mice. In 5×FAD mice, intake of WM peptide suppressed the production of inflammatory cytokines, activation of microglia, and infiltration of activated microglia around ß amyloid (Aß) depositions. WM peptide intake reduced Aß deposition in the cortex and hippocampus and then improved the object recognition memory. Taken together with previous reports, the current findings indicate that ingestion of tryptophan-related peptides or food material rich in tryptophan-related peptides, thereby regulating microglial activity, represents a potential preventive approach for cognitive decline and dementia related to inflammation.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.