Oxetanes from the ring contraction of alpha-triflates of gamma-lactones: oxetane nucleosides and oxetane amino acids

Alpha-Triflates of gamma-lactones with potassium carbonate in methanol give efficient contraction of the ring to oxetane-1-carboxylates in which the oxygen substituent at C(3) of the oxetane is predominantly trans to the carboxylate at C(2), regardless of the stereochemistry of the starting triflate. The limitations of the procedure are discussed and compared with analogous reactions for the preparation of THF carboxylates. The potential of the contraction in the preparation of oxetane nucleosides (such as oxetanocin) and oxetane sugar amino acids (analogues of oxetin) as peptidomimetics with predisposition to form secondary structural motifs is illustrated.     

Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity

Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K(m) for UDP-Glc relative to that of wild-type enzyme.     

Searching for common sequence patterns among distantly related proteins

We have developed a program Gap Allowing Pattern Explorer (GAPE)to extract amino acid sequence motifs conserved among distantlyrelated proteins. The GAPE program is designed to allow gapsin the sequences. First, this program generates all possibleamino acid patterns comprising up to five amino acids. Sequencescontaining the amino acid residues in the same order as a generatedpattern are selected as subsequences, where the differencesin the distances between two consecutive amino acids are ignored.Next, the motifs are extracted from the subsequences under conditionsin which all four distances between the five amino acids arefixed. At this stage, motifs with gaps in their subsequenceare also found by relaxing one of the four fixed distances.The statistical significance for a motif obtained is calculatedbased on the amino acid composition of the sequences under consideration.When the GAPE program was applied to 59 pyridoxal-phosphaterelatedsequences and 64 ATP (AMP-forming)-related sequences, motifsextracted with a low expectation of occurrence contained someof the amino acid residues chemically proved to be involvedin the ligand recognition.     

Exploring the Substrate Switch Motif of Aromatic Ammonia Lyases

Aromatic ammonia lyases (AALs) are important enzymes for biocatalysis as they enable the asymmetric synthesis of chiral l -α-amino acids from the corresponding α,β-unsaturated precursors. AALs have very similar protein structures and active site pockets but exhibit strict substrate specificity towards tyrosine, phenylalanine, or histidine. Herein, through systematic bioinformatics and structural analysis, we discovered eight new motifs of amino acid residues in AALs. After introducing them – as well as four already known motifs – into different AALs, we learned that altering the substrate specificity by engineering the substrate switch motif in phenylalanine ammonia lyases (PALs), phenylalanine/tyrosine ammonia lyases (PTALs), and tyrosine ammonia lyases (TALs) was only partially successful. However, we discovered that three previously unknown residue combinations introduced a substrate switch from tyrosine to phenylalanine in TAL, which was converted up to 20-fold better compared to the wild-type TAL enzyme.     

Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts

Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid-base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid-base mutant of Agrobacterium sp. beta-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid-base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.     

Growth by Insertion: The Family of Bacterial DDxP Proteins

We have identified a variety of proteins in species of the Legionella, Aeromonas, Pseudomonas, Vibrio, Nitrosomonas, Nitrosospira, Variovorax, Halomonas, and Rhizobia genera, which feature repetitive modules of different length and composition, invariably ending at the COOH side with Asp–Asp–x–Pro (DDxP) motifs. DDxP proteins range in size from 900 to 6200 aa (amino acids), and contain 1 to 5 different module types, present in one or multiple copies. We hypothesize that DDxP proteins were modeled by the action of specific endonucleases inserting DNA segments into genes encoding DDxP motifs. Target site duplications (TSDs) formed upon repair of staggered ends generated by endonuclease cleavage would explain the DDxP motifs at repeat ends. TSDs acted eventually as targets for the insertion of more modules of the same or different types. Repeat clusters plausibly resulted from amplification of both repeat and flanking TSDs. The proposed growth shown by the insertion model is supported by the identification of homologous proteins lacking repeats in Pseudomonas and Rhizobium. The 85 DDxP repeats identified in this work vary in length, and can be sorted into short (136–215 aa) and long (243–304 aa) types. Conserved Asp–Gly–Asp–Gly–Asp motifs are located 11–19 aa from the terminal DDxP motifs in all repeats, and far upstream in most long repeats.     

Extending the Scope of GTFR Glucosylation Reactions with Tosylated Substrates for Rare Sugars Synthesis

Functionalized rare sugars were synthesized with 2-, 3-, and 6-tosylated glucose derivatives as acceptor substrates by transglucosylation with sucrose and the glucansucrase GTFR from Streptococcus oralis. The 2- and 3-tosylated glucose derivatives yielded the corresponding 1,6-linked disaccharides (isomaltose analogues), whereas the 6-tosylated glucose derivatives resulted in 1,3-linked disaccharides (nigerose analogue) with high regioselectivity in up to 95 % yield. Docking studies provided insight into the binding mode of the acceptors and suggested two different orientations that were responsible for the change in regioselectivity.     

The Conversion of UDP-Glc to UDP-Man: In Silico and Biochemical Exploration To Improve the Catalytic Efficiency of CDP-Tyvelose C2-Epimerases

A promiscuous CDP-tyvelose 2-epimerase (TyvE) from Thermodesulfatator atlanticus (TaTyvE) belonging to the nucleotide sugar active short-chain dehydrogenase/reductase superfamily (NS-SDRs) was recently discovered. TaTyvE performs the slow conversion of NDP-glucose (NDP-Glc) to NDP-mannose (NDP-Man). Here, we present the sequence fingerprints that are indicative of the conversion of UDP-Glc to UDP-Man in TyvE-like enzymes based on the heptagonal box motifs. Our data-mining approach led to the identification of 11 additional TyvE-like enzymes for the conversion of UDP-Glc to UDP-Man. We characterized the top two wild-type candidates, which show a 15- and 20-fold improved catalytic efficiency, respectively, on UDP-Glc compared to TaTyvE. In addition, we present a quadruple variant of one of the identified enzymes with a 70-fold improved catalytic efficiency on UDP-Glc compared to TaTyvE. These findings could help the design of new nucleotide production pathways starting from a cheap sugar substrate like glucose or sucrose.     

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

A Simple Strategy for Glycosyltransferase‐Catalyzed Aminosugar Nucleotide Synthesis

A set of 2‐chloro‐4‐nitrophenyl glucosamino‐/xylosaminosides were synthesized and assessed as potential substrates in the context of glycosyltransferase‐catalyzed formation of the corresponding UDP/TDP‐α‐D ‐glucosamino‐/xylosaminosugars and in single‐vessel model transglycosylation reactions. This study highlights a robust platform for aminosugar nucleotide synthesis and reveals OleD Loki to be a proficient catalyst for U/TDP‐aminosugar synthesis and utilization.     

Function of lanGT3, a glycosyltransferase gene involved in landomycin A biosynthesis

The glycosyltransferase gene lanGT3, involved in the biosynthesis of the angucyclic antibiotic landomycin A, has been characterised by targeted gene deletion. A lanGT3 mutant was shown to produce landomycin E, which consists of a trisaccharide side chain attached to the polyketide moiety. Expression of lanGT3 in the mutant restored landomycin A production. Our results indicate that LanGT3 is responsible for the transfer of the fourth sugar during landomycin A biosynthesis.     

Engineering Glyco-Enzymes for Substrate Identification and Targeting

Glycoconjugates are assembled by the coordinate actions of glycosyltransferases, which add sugars, and glycosidases, which remove sugars. These glyco-enzymes comprise families of enzymes that catalyze the same reaction, making it difficult to identify the direct substrates of each isozyme. To solve this challenge, mutagenesis of glyco-enzymes has been used to enable incorporation of unnatural sugar analogs that can be employed to tag and isolate the protein substrates of an individual glycosyltransferase. A second challenge arises in efforts to determine which substrates mediate biological effects. Engineering a glycosyltransferase to target its activity toward select acceptor substrates allows deconvolution of the roles of specific glycosylation events. Similarly, glycosidases can be engineered to target specific substrates, with basic science and translational applications. This review describes recent efforts at engineering glyco-enzymes to identify and target their distinct substrates.     

Determination of Carbohydrate‐Binding Preferences of Human Galectins with Carbohydrate Microarrays

Galectins are a class of carbohydrate‐binding proteins named for their galactose‐binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate‐binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine‐specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure–activity relationships for galectin–sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation—and therefore galectin action—as either high‐affinity ligands are produced or binding is blocked. High‐affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.     

Generation of new landomycins with altered saccharide patterns through over-expression of the glycosyltransferase gene lanGT3 in the biosynthetic gene cluster of landomycin A in Streptomyces cyanogenus S-136

Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was over-expressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist of the same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results of the production of landomycin E (which contains three sugars) by the LanGT3- mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene over-expression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity of the new landomycins--compared to known ones--was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length of the deoxysugar side chain was deduced from the results.     

Inactivation of the Ketoreductase gilU Gene of the Gilvocarcin Biosynthetic Gene Cluster Yields New Analogues with Partly Improved Biological Activity

Four new analogues of the gilvocarcin‐type aryl‐C‐glycoside antitumor compounds, namely 4′‐hydroxy gilvocarcin V (4′‐OH‐GV), 4′‐hydroxy gilvocarcin M, 4′‐hydroxy gilvocarcin E and 12‐demethyl‐defucogilvocarcin V, were produced through inactivation of the gilU gene. The 4′‐OH‐analogues showed improved activity against lung cancer cell lines as compared to their parent compounds without 4′‐OH group (gilvocarcins V and E). The structures of the sugar‐containing new mutant products indicate that the enzyme GilU acts as an unusual ketoreductase involved in the biosynthesis of the C‐glycosidically linked deoxysugar moiety of the gilvocarcins. The structures of the new gilvocarcins indicate substrate flexibility of the post‐polyketide synthase modifying enzymes, particularly the C‐glycosyltransferase and the enzyme responsible for the sugar ring contraction. The results also shed light into biosynthetic sequence of events in the late steps of biosynthetic pathway of gilvocarcin V.     

Scalable and Selective β-Hydroxy-α-Amino Acid Synthesis Catalyzed by Promiscuous l-Threonine Transaldolase ObiH

Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well-characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β-hydroxy-α-amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an l -threonine transaldolase, to achieve selective milligram-scale synthesis of a diverse array of non-standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re-entry into the catalytic cycle. ObiH-catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β-hydroxy-α-amino acids possessing broad functional-group diversity. Furthermore, we demonstrated that ObiH-generated β-hydroxy-α-amino acids could be modified through additional transformations to access important motifs, such as β-chloro-α-amino acids and substituted α-keto acids.     

LanGT2 Catalyzes the First Glycosylation Step during landomycin A biosynthesis

The glycosyltransferase LanGT2 is involved in the biosynthesis of the hexasaccharide side chain of the angucyclic antibiotic landomycin A. Its function was elucidated by targeted gene inactivation of lanGT2. The main metabolite of the obtained mutant was identified as tetrangulol (4), the progenitor of the landomycin aglycon (7). The lack of the sugar side chain indicates that LanGT2 catalyzes the priming glycosyl transfer in the hexasaccharide biosynthesis: the attachment of a D-olivose to O-8 of the polyketide backbone. Heterologous expression of urdGT2 from S. fradiae Tü2717 in this mutant resulted in the production of a novel C-glycosylated angucycline (6).     

Pyruvate-Kinase-Coupled Glycosyltransferase Assays: Limitations,Struggles and Problem Resolution

Enzyme assays involving coupled pyruvate kinase (PK) have been used for many years to monitor the activity of major classes of enzymes including glycosyltransferases. Numerous potent inhibitors have been discovered and kinetically characterized thanks to this technology. However, when inhibitors of these important enzymes are screened, PK inhibitors or activators are very often observed. In this study we report solutions to resolve the problems encountered either during the screening or during the kinetic characterization of glycosyltransferase inhibitors by means of PK-coupled assays. The enzyme under study—WaaC—is an important glycosyltransferase involved in the bacterial lipopolysaccharide (LPS) biosynthesis pathway. Firstly we showed that alternative kinases such as nucleoside 5-diphosphate kinase (NDPK), myokinase (MK), and ADPdependent hexokinase that catalyze similar reactions to PK are prone to the same troubles. Moreover, an ADP chemosensor was used as an alternative but the sensitivity was not sufficient to allow a proper screening. Finally, we found that a stepwise PK/luciferase assay resolved the problems encountered with PK inhibitors and that a WaaC HPLC assay allowed the identification of WaaC inhibitors acting as PK activators, thus allowing false positive and false negative results linked to the coupling to PK to be eliminated.     

WideEffHunter: An Algorithm to Predict Canonical and Non-Canonical Effectors in Fungi and Oomycetes

Newer effectorome prediction algorithms are considering effectors that may not comply with the canonical characteristics of small, secreted, cysteine-rich proteins. The use of effector-related motifs and domains is an emerging strategy for effector identification, but its use has been limited to individual species, whether oomycete or fungal, and certain domains and motifs have only been associated with one or the other. The use of these strategies is important for the identification of novel, non-canonical effectors (NCEs) which we have found to constitute approximately 90% of the effectoromes. We produced an algorithm in Bash called WideEffHunter that is founded on integrating three key characteristics: the presence of effector motifs, effector domains and homology to validated existing effectors. Interestingly, we found similar numbers of effectors with motifs and domains within two different taxonomic kingdoms: fungi and oomycetes, indicating that with respect to their effector content, the two organisms may be more similar than previously believed. WideEffHunter can identify the entire effectorome (non-canonical and canonical effectors) of oomycetes and fungi whether pathogenic or non-pathogenic, unifying effector prediction in these two kingdoms as well as the two different lifestyles. The elucidation of complete effectoromes is a crucial step towards advancing effectoromics and disease management in agriculture.