Glycosynthase activity of Geobacillus stearothermophilus GH52 beta-xylosidase: efficient synthesis of xylooligosaccharides from alpha-D-xylopyranosyl fluoride through a conjugated reaction

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of alpha-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K(M(acceptor)), respectively, and a k(cat)/K(M(acceptor)) value of 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.     

Characterization of a Galactosynthase Derived from Bacillus circulans β‐Galactosidase: Facile Synthesis of D‐Lacto‐ and D‐Galacto‐N‐bioside

Glycosynthases—retaining glycosidases mutated at their catalytic nucleophile—catalyze the formation of glycosidic bonds from glycosyl fluorides as donor sugars and various glycosides as acceptor sugars. Here the first glycosynthase derived from a family 35 β‐galactosidase is described. The Glu→Gly mutant of BgaC from Bacillus circulans (BgaC‐E233G) catalyzed regioselective galactosylation at the 3‐position of the sugar acceptors with α‐galactosyl fluoride as the donor. Transfer to 4‐nitophenyl α‐D ‐N‐acetyl‐glucosaminide and α‐D ‐N‐acetylgalactosaminide yielded 4‐nitophenyl α‐lacto‐N‐biose and α‐galacto‐N‐biose, respectively, in high yields (up to 98 %). Kinetic analysis revealed that the high affinity of the acceptors contributed mostly to the BgaC‐E233G‐catalyzed transglycosylation. BgaC‐E233G showed no activity with β‐(1,3)‐linked disaccharides as acceptors, thus suggesting that this enzyme can be used in “one‐pot synthesis” of LNB‐ or GNB‐containing glycans.     

Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.     

Highly productive autocondensation and transglycosylation reactions with Sulfolobus solfataricus glycosynthase

Transglycosylation reactions (autocondensation of the substrate or transfer of the glycon donor moiety to different acceptors) with the hyperthermophilic glycosynthase from Sulfolobus solfataricus acting in dilute sodium formate buffer at pH 4.0 are reported; the use of 4-nitrophenyl beta-glucopyranoside as both donor and acceptor in the self-transfer reaction and a highly productive reaction with 1.1 M 2-nitrophenyl beta-glucopyranoside were possible. Interesting effects, governed by the anomeric configuration and lipophilicity of heteroacceptors, on the regioselectivity and yield of reactions were found for the first time with this enzyme and are discussed. The results demonstrate the unexplored synthetic potential of this glycosynthase; the tuning of the reaction conditions and the choice of different donors/acceptors can lead to products of applicative interest.     

Site-directed mutagenesis at the active site Trpl20 of Aspergillus awamori glucoamylase

Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (k_cat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (K_M) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 – Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 – 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 – Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2.     

Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium

Highly branched fatty acids, the main components of the preen-gland waxes of the domestic goose and the Muscovy duck, and their derivatives are promising chiral precursors for the synthesis of macrolide antibiotics. The key step in the utilisation of these compounds is their regioselective hydroxylation, which cannot be achieved in a classical chemical approach. Three P450 monooxygenases, CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in the hydroxylation of iso and anteiso fatty acids (>400 min(-1)), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium and its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases, the triple mutant showed much higher activities than the wild-type enzyme. The binding constants, determined for wild-type CYP102A1 and the triple mutant with tetramethylnonanol as substrate, were >200 microM and approximately 23 microM, respectively. Data derived from binding analysis support the differences in activity found for the wild-type CYP102A1 and the triple mutant. Surprisingly, CYP102A2 and CYP102A3 from Bacillus subtilis did not show any activity. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme's active sites, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not come into proximity with the heme.     

Modification of S1 subsite specificity in the cysteine protease cathepsin B

Cysteine proteases of the papain family generally exhibit broadP₁ specificity. A notable exception is papaya proteinase IV(PPIV), which only accepts Gly at this position. In all othercysteine proteases the S₁ subsite residues 23 and 65 (papainnumbering) are absolutely conserved as Gly, while in PPIV theyare replaced by Glu and Arg, respectively. These differencesappear to underlie both PPIV specificity and its resistanceto inhibition by cystatins. To test this hypothesis, the equivalentresidues (Gly27 and Gly73) in the mammalian cysteine proteasecathepsin B were changed to Glu and Arg, respectively. Relativeto the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showeda drastic reduction in activity with substrates containing aP₁ Arg. In contrast, substrates having a Gly residue in P₁ werehydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg)exhibited no detectable activity against any substrate studied.Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane]was found to be similar to that of the wild-type enzyme. Incontrast, inhibition by cystatin C exhibited a 20 000-fold reduction.These results demonstrate the dramatic influence of side chainsat sequence locations 27 and 73 on the S₁ subsite specificityof cysteine proteases.     

1,3-二氧戊环-2-苯基-4,5-二苄溴的合成

以酒石酸为原料通过改进的酯化方法合成了酒石酸二甲酯,经苯甲醛缩合,AlLiH4还原,磺酰化,以及使用LiBr进行溴代最终制得标题化合物.     

双对二甲氨基苯甲醛缩1,4-二(3-氨基丙基)哌嗪希夫碱的合成工艺及抑菌活性研究

以对二甲氨基苯甲醛(DMAB)和1,4-二(3-氨基丙基)哌嗪(BAPP)为原料,通过缩合反应合成了一个新的DMAB-BAPP希夫碱.利用正交试验确定了最佳合成工艺条件,即反应温度50℃,反应时间4h,DMAB与BAPP投料摩尔比是2.1∶1,在此条件下,产物收率为83.7％.通过琼脂扩散法测定了产物的抑菌性能,结果表...     

Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose–acceptor reaction—as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.     

Acetals and Ethers. XVIII. Reaction products of prop-2-enal and but-2-enal with mixture: n-Aliphatic alcohol and ethylene glycol

The direct condensation reaction of prop-2-enal or but-2-enal with mixture of n-aliphatic alcohol and ethylene glycol, in the presence of p-toluenesulphonic acid as catalyst, leads to a complex mixture of saturated, unsaturated, cyclic and linear acetals, moreover, 2-(2-alkoxy-alkyl)-1,3-dioxolanes are the main reaction products. The detailed investigations for n-butanol showed that unsaturated cyclic acetals: 2-vinyl-1,2-dioxolane 1a or 2-(1-propenyl)-1,3-dioxolane 1b , as well as unsaturated linear acetals: 1,1-dibutoxy-prop-2-en 2a or 1,1-dibutoxy-but-2-en 2b are intermediate reaction products. Additionally, it was found in final products presence of eight by-products: 5-butoxy- 4a or 5-butoxy-7-methyl-1,4-dioxepane 4b , 1,1,3-tributoxypropane 5a or 1,1,3-tributoxybutane 5b , 2-[2-(2-hydroxyethoxy)ethyl]- 6a or 2-[2-(2-hydroxyethoxy)propyl]-1,3-dioxolane 6b , 5-(2-hydroxyethoxy)-7-methyl-1,4-dioxepane 7b , 1,3-dibutoxy-1-(2-hydroxyethoxy)-propane 8a or 1,3-dibutoxy-1-(2-hydroxyethoxy)-butane 8b , 1,1-dibutoxy-3-(2-hydroxyethoxy)-propane 9a or 1,1-dibutoxy-3-(2-hydroxyethoxy)-butane 9b , 3-butoxy-1,1-bis-(2-hydroxyethoxy)-propane 10a or 3-butoxy-1,1-bis-(2-hydroxyethoxy)-butane 10b , and 1-butoxy-1,3-bis-(2-hydroxyethoxy)-propane 11a or 1-butoxy-1,3-bis-(2-hydroxyethoxy)-butane 11b , respectively.     

The effect of aromatic diboronic acid on characteristics of polybenzoxazine based on phenol and 4-aminomethylbenzoate

In this study, a polybenzoxazine, (PPab) based on phenol and 4-aminomethyl benzoate and its composites containing various amounts of 1,4-benzene diboronic acid, (BDBA) were synthesized by stepwise curing. It has been determined that both thermal stability and char yield were increased as the amount of BDBA incorporated was increased. The improvements in thermal characteristics were associated with condensation reactions of B-OH of BDBA and not only with hydroxyl groups of polybenzoxazine but also ester groups on the aniline linkages yielding a highly cross-linked structure. In addition, self-condensation reactions of B-OH groups generated boroxine net-work. Strong evidences for growth of this net-work on benzoate units of the polymer were detected.     

Synthese von 2-alkoxysubstituierten Oligo- und Poly(1,4-phenylenethenylen)en und 2-Arylbenzo[b]furanen mit Hilfe der Siegrist-Reaktion

Synthesis of 2-Alkoxy Substituted Oligo- and Poly(1,4-phenyleneethylene)s and 2-Arylbenzo[b]furanes by Applying the Siegrist Reaction Alkylation of 2-hydroxy-4-methylbenzaldehyde ( 1 ) yields the 2-alkoxy-4-methylbenzaldehydes ( 2a-l ) which can be easily transformed to the Schiff bases 3a-l . The intermolecular self-condensation in a strongly alkaline medium leads to the oligo- and poly(1,4-phenyleneethenylene)s ( 4a-i ) with an outstanding regular constitution and overall (E)-configuration. The terminal N-arylamino group can be cleaved by hydrolysis generating the compounds 5a-i . An intramolecular condensation forming the benzo[b]furanes 6j,k is observed for 3j,k – due to the activated OCH₂ group in 2-position. Finally, both the 4-CH₃ as well as the 2-OCH₂ group take part in the reaction of 3l . The twofold Schiff base 3m , obtained from 1 via 2m , yields the ladder polymer 7m .     

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala

The 3-D structure of subtilisin YaB was computer modelled using the structures of subtilisin BPN', subtilisin Carlsberg and thermitase as templates. Gly124 and Gly151 located on both sides of the waist of the S1 pocket were selected for site-directed mutagenesis based on the modelled structure. The mutated ale genes coding for the mutant subtilisin YaB were expressed in Bacillus subtilis DB104. All of the G124 and G151 series of mutants exhibited an increase of relative catalytic activity for elastin-orcein against casein and myofibrillar proteins. The S1 substrate specificity of G124A, G124V and G151A mutants were assessed using various carbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro or Val)-(Ala, Phe or Leu)-p- nitroanilide [AA(P/V) (A/F/L)]. While G124A and G124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzed Ala, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPF and AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Km values approximately 3-10-fold higher than those of the wild-type enzyme. The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVA with kcat/Km values approximately 1-4-fold higher than those of the wild-type enzyme. These results clearly indicate that the S1 substrate specificity of G124A and G124V mutants was restricted to Ala and Gly, and G151A mutant to Ala, Gly and Leu.      

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.     

Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.     

The STOBBE condensation. X. The cyclisation of (E)-3-methoxycarbonyl-4-(5′-methyl-2′-furyl)-but-3-enoic acid,and (E)−3-methoxycarbonyl-4-(2′-furyl)-pent-3-enoic acid,to Benzofuran Derivatives

The condensation of 5-methyl-furan-2-aldehyde and 2-furyl-methyl ketone with dimethyl succinate using either potassium t-butoxide or sodium hydride as condensing agents, gives predominantly (E)-3-methoxycarbonyl-4-(5′-methyl-2′-furyl)-but-3-enoic acid 1a and (E)-3-methoxy-carbonyl-4-(2′furyl)-pent-3-enoic acid 5 respectively. Their configurations are inferred by cyclisation with sodium acetate in acetic anhydride to the corresponding benzofuran derivatives 2,6 . Alcoholysis of (E)-3-carboxy-4-(5′-methyl-2′-furyl)-but-3-enoic anhydride 3 gives the half-ester 1c which is isomeric with the half-ester 1a . A competing side reaction also gives the self-condensation product of the succinic ester 4 .     

Directed evolution of a β-glycosidase from Agrobacterium sp. to enhance its glycosynthase activity toward C3-modified donor sugars

Glycans bearing modified hydroxyl groups are common in biology but because these modifications are added after assembly, enzymes are not available for the transfer and coupling of hydroxyl-modified monosaccharide units. Access to such enzymes could be valuable, particularly if they can also introduce 'bio-orthogonal tags'. Glycosynthases, mutant glycosidases that synthesize glycosides using glycosyl fluoride donors, are a promising starting point for creation of such enzymes through directed evolution. Inspection of the active site of a homology model of the GH1 Agrobacterium sp. β-glycosidase, which has both glucosidase and galactosidase activity, identified Q24, H125, W126, W404, E411 and W412 as amino acids that constrain binding around the 3-OH group, suggesting these residues as targets for mutation to generate an enzyme capable of handling 3-O-methylated sugars. Site-directed saturation mutagenesis at these positions within the wild-type β-glycosidase gene and screening via an on-plate assay yielded two mutants (Q24S/W404L and Q24N/W404N) with an improved ability to hydrolyze 4-nitrophenyl 3-O-methyl-β-d-galactopyranoside (3-MeOGal-pNP). Translation of these mutations into the evolved glycosynthase derived from the same glucosidase (2F6) yielded glycosynthases (AbgSL-T and AbgNN-T, where T denotes transferase) capable of forming 3-O-methylated glucosides on multi-milligram scales at rates approximately 5 and 40 times greater, respectively, than the parent glycosynthase.     

The Multivalent Polyampholyte Domain of Nst1, a P-Body-Associated Saccharomyces cerevisiae Protein,Provides a Platform for Interacting with P-Body Components

The condensation of nuclear promyelocytic leukemia bodies, cytoplasmic P-granules, P-bodies (PBs), and stress granules is reversible and dynamic via liquid–liquid phase separation. Although each condensate comprises hundreds of proteins with promiscuous interactions, a few key scaffold proteins are required. Essential scaffold domain sequence elements, such as poly-Q, low-complexity regions, oligomerizing domains, and RNA-binding domains, have been evaluated to understand their roles in biomolecular condensation processes. However, the underlying mechanisms remain unclear. We analyzed Nst1, a PB-associated protein that can intrinsically induce PB component condensations when overexpressed. Various Nst1 domain deletion mutants with unique sequence distributions, including intrinsically disordered regions (IDRs) and aggregation-prone regions, were constructed based on structural predictions. The overexpression of Nst1 deletion mutants lacking the aggregation-prone domain (APD) significantly inhibited self-condensation, implicating APD as an oligomerizing domain promoting self-condensation. Remarkably, cells overexpressing the Nst1 deletion mutant of the polyampholyte domain (PD) in the IDR region (Nst1_∆PD) rarely accumulate endogenous enhanced green fluorescent protein (EGFP)-tagged Dcp2. However, Nst1_∆PD formed self-condensates, suggesting that Nst1 requires PD to interact with Dcp2, regardless of its self-condensation. In Nst1_∆PD-overexpressing cells treated with cycloheximide (CHX), Dcp2, Xrn1, Dhh1, and Edc3 had significantly diminished condensation compared to those in CHX-treated Nst1-overexpressing cells. These observations suggest that the PD of the IDR in Nst1 functions as a hub domain interacting with other PB components.     

A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases

Carboxylesterases containing the sequence motif GGGX catalyze the hydrolysis of esters of chiral tertiary alcohols, albeit with only low to moderate enantioselectivity, for three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity for this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this amino acid for an alanine residue led to a sixfold increase of enantioselectivity (E = 19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is specific: the same mutant had the opposite enantiopreference towards the substrate linalyl acetate. Thus, depending on the substrate structure, the same mutant has either increased enantioselectivity or opposite enantiopreference compared to the wild-type enzyme.