Purification and characterization of an acetyl xylan esterase from Bacillus pumilus. (1/152)

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.  (+info)

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase. (2/152)

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.  (+info)

Lysosomal and cytosolic sialic acid 9-O-acetylesterase activities can Be encoded by one gene via differential usage of a signal peptide-encoding exon at the N terminus. (3/152)

9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5' region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5'-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters.  (+info)

Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. (4/152)

A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp. strain PC-2. The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da. An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli. Two active forms of the esterase were purified from recombinant E. coli cultures. The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide. The enzyme released acetate from several acetylated substrates, including acetylated xylan. The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus. Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1). It had temperature and pH optima of 30 degrees C and 9.0, respectively. AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA. The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum. The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex. Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.  (+info)

Acetylation: a regulatory modification to rival phosphorylation? (5/152)

The fact that histones are modified by acetylation has been known for almost 30 years. The recent identification of enzymes that regulate histone acetylation has revealed a broader use of this modification than was suspected previously. Acetylases are now known to modify a variety of proteins, including transcription factors, nuclear import factors and alpha-tubulin. Acetylation regulates many diverse functions, including DNA recognition, protein-protein interaction and protein stability. There is even a conserved structure, the bromodomain, that recognizes acetylated residues and may serve as a signalling domain. If you think all this sounds familiar, it should be. These are features characteristic of kinases. So, is acetylation a modification analogous to phosphorylation? This review sets out what we know about the broader substrate specificity and regulation of acetyl- ases and goes on to compare acetylation with the process of phosphorylation.  (+info)

Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. (6/152)

BACKGROUND: The complex polysaccharide rhamnogalacturonan constitutes a major part of the hairy region of pectin. It can have different types of carbohydrate sidechains attached to the rhamnose residues in the backbone of alternating rhamnose and galacturonic acid residues; the galacturonic acid residues can be methylated or acetylated. Aspergillus aculeatus produces enzymes that are able to perform a synergistic degradation of rhamnogalacturonan. The deacetylation of the backbone by rhamnogalacturonan acetylesterase (RGAE) is an essential prerequisite for the subsequent action of the enzymes that cleave the glycosidic bonds. RESULTS: The structure of RGAE has been determined at 1.55 A resolution. RGAE folds into an alpha/beta/alpha structure. The active site of RGAE is an open cleft containing a serine-histidine-aspartic acid catalytic triad. The position of the three residues relative to the central parallel beta sheet and the lack of the nucleophilic elbow motif found in structures possessing the alpha/beta hydrolase fold show that RGAE does not belong to the alpha/beta hydrolase family. CONCLUSIONS: Structural and sequence comparisons have revealed that, despite very low sequence similarities, RGAE is related to seven other proteins. They are all members of a new hydrolase family, the SGNH-hydrolase family, which includes the carbohydrate esterase family 12 as a distinct subfamily. The SGNH-hydrolase family is characterised by having four conserved blocks of residues, each with one completely conserved residue; serine, glycine, asparagine and histidine, respectively. Each of the four residues plays a role in the catalytic function.  (+info)

Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences. (7/152)

Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.  (+info)

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity. (8/152)

The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.  (+info)

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