Enzymatic Synthesis of Novel Vitexin Glucosides

Vitexin is a C-glucoside flavone that exhibits a wide range of pharmaceutical activities. However, the poor solubility of vitexin limits its applications. To resolve this limitation, two glycoside hydrolases (GHs) and four glycosyltransferases (GTs) were assayed for glycosylation activity toward vitexin. The results showed that BtGT_16345 from the Bacillus thuringiensis GA A07 strain possessed the highest glycosylation activity, catalyzing the conversion of vitexin into new compounds, vitexin-4′-O-β-glucoside (1) and vitexin-5-O-β-glucoside (2), which showed greater aqueous solubility than vitexin. To our knowledge, this is the first report of vitexin glycosylation. Based on the multiple bioactivities of vitexin, the two highly soluble vitexin derivatives might have high potential for pharmacological usage in the future.     

Novel Carboxyl Esterase Preparations for the Resolution of Linalyl Acetate

Summary.  Biocatalytic resolution of the tertiary terpene alcohol (±)-linalool was accomplished via hydrolysis of its corresponding acetate ester using two highly enantiospecific enzymes (E > 100). The latter were identified in a crude cell-free extract of Rhodococcus ruber DSM 43338 and could be separated by (partial) protein purification. Since they showed opposite enantiopreference, they were termed (R)- and (S)-linalyl acetate hydrolase (LAH). The activity and selectivity of the enzyme preparations was markedly dependent on the fermentation conditions. Received November 18, 1999. Accepted January 21, 2000     

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear β-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA²Xyl_n. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.     

Elucidating Sequence and Structural Determinants of Carbohydrate Esterases for Complete Deacetylation of Substituted Xylans

Acetylated glucuronoxylan is one of the most common types of hemicellulose in nature. The structure is formed by a β-(1→4)-linked D-xylopyranosyl (Xylp) backbone that can be substituted with an acetyl group at O-2 and O-3 positions, and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Acetyl xylan esterases (AcXE) that target mono- or doubly acetylated Xylp are well characterized; however, the previously studied AcXE from Flavobacterium johnsoniae (FjoAcXE) was the first to remove the acetyl group from 2-O-MeGlcpA-3-O-acetyl-substituted Xylp units, yet structural characteristics of these enzymes remain unspecified. Here, six homologs of FjoAcXE were produced and three crystal structures of the enzymes were solved. Two of them are complex structures, one with bound MeGlcpA and another with acetate. All homologs were confirmed to release acetate from 2-O-MeGlcpA-3-O-acetyl-substituted xylan, and the crystal structures point to key structural elements that might serve as defining features of this unclassified carbohydrate esterase family. Enzymes comprised two domains: N-terminal CBM domain and a C-terminal SGNH domain. In FjoAcXE and all studied homologs, the sequence motif around the catalytic serine is Gly-Asn-Ser-Ile (GNSI), which differs from other SGNH hydrolases. Binding by the MeGlcpA-Xylp ligand is directed by positively charged and highly conserved residues at the interface of the CBM and SGNH domains of the enzyme.     

巨大芽胞杆菌环氧水解酶的固定化及其催化性能

考察了多种载体对巨大芽孢杆菌ECU1001环氧水解酶的固定化.以大孔DEAE-纤维素离子交换树脂为载体时,固定化酶的活力回收达70%.进一步考察了温度和pH对固定化酶活力的影响,并使用该固定化酶进行了缩水甘油苯基醚对映选择性水解批次反应.结果表明,在较低的底物浓度下该固定化酶的稳定性较好,10批反应后仍然剩余72.4%的活力.     

Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved α,β-hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the α,β-hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.     

脂肪酰胺水解酶催化三联体膦酰化失活的机理：一个模型体系的理论研究

甲基花生四烯基氟代膦酸酯(MAFP)是脂肪酰胺水解酶(FAAH)的一个抑制剂. FAAH的丝氨酸241(Ser241)-丝氨酸217(Ser217)-赖氨酸142(Lys142)催化三联体被MAFP膦酰化后将导致FAAH失活. 本文采用B3LYP/6-311G(d,p)和MP2/6-311G(d,p)方法及一个简化的计算模型体系对这个膦酰化抑制反应进行理论研究. 考虑了两种反应途径. Path A涉及FAAH的催化三联体的所有残基, 是一个分步的加成-消除过程, 形成两性离子的三角双锥中间体, 其中第一步反应是决速步骤. 在这个反应途径中, Ser217和Lys142对亲核试剂Ser241起到碱催化活化的作用, 而Ser217充作Lys142和Ser241之间的桥梁. 此外, 溶剂中的一个水分子作为Lys142和MAFP间的“氢桥”具有关键的作用, 通过给出和接收质子促进了长距离的质子转移. Path B是催化三联体中的残基Lys142被突变为丙氨酸以后的膦酰化反应, 也是一个分步过程. 水的本体溶剂效应通过极化连续介质模型(PCM)估算. 计算结果显示膦酰化反应的Path A是优势途径, 在水溶液中其决速步骤的活化能垒为64.9 kJ·mol-1. FAAH催化三联体中残基Lys142的变异会降低膦酰化反应的速率, 这与实验结果相一致.     

Theoretical study of the full reaction mechanism of human soluble epoxide hydrolase

The complete reaction mechanism of soluble epoxide hydrolase (sEH) has been investigated by using the B3LYP density functional theory method. Epoxide hydrolases catalyze the conversion of epoxides to their corresponding vicinal diols. In our theoretical study, the sEH active site is represented by quantum-chemical models that are based on the X-ray crystal structure of human soluble epoxide hydrolase. The trans-substituted epoxide (1S,2S)-beta-methylstyrene oxide has been used as a substrate in the theoretical investigation of the sEH reaction mechanism. Both the alkylation and the hydrolytic half-reactions have been studied in detail. We present the energetics of the reaction mechanism as well as the optimized intermediates and transition-state structures. Full potential energy curves for the reactions involving nucleophilic attack at either the benzylic or the homo-benzylic carbon atom of (1S,2S)-beta-methylstyrene oxide have been computed. The regioselectivity of epoxide opening has been addressed for the two substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide.     

Hydrolysis of BNPP Catalyzed by the Crowned Schiff Base Co(II) Complex Containing Benzoaza‐15‐Crown‐5 in Micellar Solution

It has been reported that three aza crowned Schiff base cobalt (II) complexes were synthesized and characterized, and the metallomicelle made up of the cobalt (II) complexes and surfactants(Brij35, CTAB, LSS), as mimic hydrolytic metalloenzyme, was used in catalytic hydrolysis of bis(4‐nitrophenyl) phosphate (BNPP). The analysis of specific absorption spectrums of the hydrolytic reaction systems indicated that key intermediates made up of BNPP and Co (II) complexes are formed in the reaction process of BNPP catalytic hydrolysis. The mechanism of BNPP catalytic hydrolysis proposed is based on the analytic result of specific absorption spectra. Based on the mechanism proposed, a kinetic mathematical model for the calculation of the kinetic parameter of BNPP catalytic hydrolysis has been established. The acid effect of reaction system, structure effect of the complexes, effect of temperature and effect of micelles on the rate of BNPP hydrolysis catalyzed by the complexes have been discussed.