Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus

Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan‐degrading organism whose genome encodes a large number of open reading frames annotated as fiber‐degrading enzymes. We have determined the three‐dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains—a C‐terminal SGNH domain and an N‐terminal jelly‐roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para‐nitrophenyl acetate and para‐nitrophenyl butyrate. The suite of fiber‐degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short‐chain acyl esters (C2–C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4‐nitrophenyl‐β‐D ‐xylopyranoside monoacetates as substrates in a β‐xylosidase‐coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine‐substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β‐hydrolase fold. TM0077 assembles into a doughnut‐shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Structural and functional study of FK domain of Fstl1

Fstl1 is a TGF‐β superfamily binding protein which involved in many pathological processes. The function of Fstl1 has been widely elucidated, but its structural characterization has not been explored. Here we solved the high‐resolution crystal structure of FK domain of murine Fstl1, analyzed its unique characteristics, and investigated its contribution to the function of full‐length Fstl1. We found that Fstl1‐FK forms a stable dimer in both solution and crystal, which suggest that this protein may function as a dimer during its interaction with TGF‐β, a molecule known to form dimer during activation process. We also found this FK domain is indispensable for the proper function of Fstl1 during the transduction of TGF‐β signaling. These observations provide important insights into the understanding of Fstl1 and may facilitate the exploration of this molecule in clinical study.     

Hydroxynitrile lyase from Hevea brasiliensis: Molecular characterization and mechanism of enzyme catalysis

(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C(SINGLE BOND)C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the α/β hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of the predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis. Proteins 27:438–449, 1997. © 1997 Wiley-Liss, Inc.     

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.     

Crystallographic and mutational analyses of tannase from Lactobacillus plantarum

Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of α/β‐hydrolase superfamily with an additional “lid” domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate. Proteins 2013; 81:2052–2058. © 2013 Wiley Periodicals, Inc.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

Structural and functional characterization of a novel acidophilic 7α‐hydroxysteroid dehydrogenase

7α‐Hydroxysteroid dehydrogenase (7α‐HSDH) is an NAD(P)H‐dependent oxidoreductase belonging to the short‐chain dehydrogenases/reductases. In vitro, 7α‐HSDH is involved in the efficient biotransformation of taurochenodeoxycholic acid (TCDCA) to tauroursodeoxycholic acid (TUDCA). In this study, a gene encoding novel 7α‐HSDH (named as St‐2‐1) from fecal samples of black bear was cloned and heterologously expressed in Escherichia coli. The protein has subunits of 28.3 kDa and a native size of 56.6 kDa, which suggested a homodimer. We studied the relevant properties of the enzyme, including the optimum pH, optimum temperature, thermal stability, activators, and inhibitors. Interestingly, the data showed that St‐2‐1 differs from the 7α‐HSDHs reported in the literature, as it functions under acidic conditions. The enzyme displayed its optimal activity at pH 5.5 (TCDCA). The acidophilic nature of 7α‐HSDH expands its application environment and the natural enzyme bank of HSDHs, providing a promising candidate enzyme for the biosynthesis of TUDCA or other related chemical entities.     

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α‐hydroxyl group. The rate‐determining enzyme in this pathway is bile acid 7α‐dehydratase (baiE). In this study, crystal structures of apo‐BaiE and its putative product‐bound [3‐oxo‐Δ^4,6‐lithocholyl‐Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site‐directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady‐state kinetic studies reveal that the BaiE homologs are able to turn over 3‐oxo‐Δ⁴‐bile acid and CoA‐conjugated 3‐oxo‐Δ⁴‐bile acid substrates with comparable efficiency questioning the role of CoA‐conjugation in the bile acid metabolism pathway. Proteins 2016; 84:316–331. © 2016 Wiley Periodicals, Inc.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Structural and biochemical analyses of YvgN and YtbE from Bacillus subtilis

Bacillus subtilis is one of the most studied gram‐positive bacteria. In this work, YvgN and YtbE from B. subtilis, assigned as AKR5G1 and AKR5G2 of aldo‐keto reductase (AKR) superfamily. AKR catalyzes the NADPH‐dependent reduction of aldehyde or aldose substrates to alcohols. YvgN and YtbE were studied by crystallographic and enzymatic analyses. The apo structures of these proteins were determined by molecular replacement, and the structure of holoenzyme YvgN with NADPH was also solved, revealing the conformational changes upon cofactor binding. Our biochemical data suggest both YvgN and YtbE have preferential specificity for derivatives of benzaldehyde, such as nitryl or halogen group substitution at the 2 or 4 positions. These proteins also showed broad catalytic activity on many standard substrates of AKR, such as glyoxal, dihydroxyacetone, and DL‐glyceraldehyde, suggesting a possible role in bacterial detoxification.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and characterization of β-agarase from agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg⁻¹ protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg²⁺, Ag⁺ and Cu²⁺, whereas 25–40% of native activity was retained in the presence of Zn²⁺, Sn²⁺ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.     

Structural characterization of neutral and anionic glucans from Mesorhizobium loti

The periplasmic glucans of Mesorhizobium loti were isolated and separated into fractions according to their acidity. NMR spectroscopy confirmed their backbone structure to be a cyclic beta-(1-->2)-d-glucan as in the case of other rhizobia, and revealed no non-glycosidic substituents in the neutral fraction, and glycerophosphoryl and succinyl residues as major and minor substituents, respectively, in the anionic fractions. MALDI-TOF mass spectrometry showed that the anionic glucans contain one, two, or three such substituents per molecule according to their acidity, and, in contrast, that all the anionic subfractions have a similar size distribution to that of the neutral glucans, where molecules composed of 20-24 glucosyl residues are predominant. These results clarify the periplasmic glucan composition in terms of charge-to-mass ratios in M. loti cells.