Crystal structure of Bacillus sp. GL1 xanthan lyase complexed with a substrate: insights into the enzyme reaction mechanism

Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8 (PL-8), acts exolytically on the side-chains of pentasaccharide-repeating polysaccharide xanthan and cleaves the glycosidic bond between glucuronic acid (GlcUA) and pyruvylated mannose (PyrMan) through a beta-elimination reaction. To clarify the enzyme reaction mechanism, i.e. its substrate recognition and catalytic reaction, we determined crystal structures of a mutant enzyme, N194A, in complexes with the product (PyrMan) and a substrate (pentasacharide) and in a ligand-free form at 1.8, 2.1, and 2.3A resolution. Based on the structures of the mutant in complexes with the product and substrate, we found that xanthan lyase recognized the PyrMan residue at subsite -1 and the GlcUA residue at +1 on the xanthan side-chain and underwent little interaction with the main chain of the polysaccharide. The structure of the mutant-substrate complex also showed that the hydroxyl group of Tyr255 was close to both the C-5 atom of the GlcUA residue and the oxygen atom of the glycosidic bond to be cleaved, suggesting that Tyr255 likely acts as a general base that extracts the proton from C-5 of the GlcUA residue and as a general acid that donates the proton to the glycosidic bond. A structural comparison of catalytic centers of PL-8 lyases indicated that the catalytic reaction mechanism is shared by all members of the family PL-8, while the substrate recognition mechanism differs.     

Characterization of the glucosyltransferases that assemble the side chains of the Indian Leishmania donovani lipophosphoglycan

The bacterium Bacillus sp. GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products. A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells. The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859. The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C. Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases.     

C-S lyase activities in leaves of crucifers and non-crucifers, and the characterization of three classes of C-S lyase activities from oilseed rape (Brassica napus L.)

C-S lyases in plants are involved in primary and secondary metabolism, and in glucosinolate-containing species may be involved in glucosinolate biosynthesis. Extracts from oilseed rape ( Brassica napus ) leaves were assayed for several C-S lyase activities. Four activities [using L -cystine, L -cystathionine, S -(2-benzothiazolyl)- L -Cys (SBC) and S -benzyl- L -Cys] were investigated in detail. All are developmentally regulated (highest in youngest leaves), and differentially inhibited by iodoacetamide, N -ethylmaleimide (NEM) and ethylenediaminetetraacetic acid (EDTA). Thermal stabilities and pH optima were also distinct. Competitive inhibition of the SBC lyase activity with a variety of sulphur-containing compounds indicated that cystine lyase contributes to SBC degradation, and this enzyme may cleave a wide range of compounds, both aliphatic and aromatic, but other 'SBC lyases' were also present. Putative aromatic glucosinolate intermediates were cleaved by the rape enzymes. Developmental and biochemical studies indicate that at least three classes of C-S lyase activity are present in rape leaves: cystathionine β -lyase, cystine lyase and a group of relatively non-specific lyases. C-S lyase preparations from other glucosinolate- and non-glucosinolate-containing species were capable of cleaving a number of aliphatic and aromatic conjugates. The highest activities were detected in glucosinolate-containing species and Allium cepa (onion). C-S lyase activities in non-glucosinolate-containing species (tobacco, Nicotiana tabacum, and barley, Hordeum vulgare ) were much lower.     

Crystal structure of Bacillus sp. GL1 xanthan lyase,which acts on the side chains of xanthan

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 A resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal alpha-helical and C-terminal beta-sheet domains, which constitute incomplete alpha(5)/alpha(5)-barrel and anti-parallel beta-sheet structures, respectively. A deep cleft is located in the N-terminal alpha-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg(313) and Tyr(315) residues in the loop from the N-terminal domain and the Arg(612) residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.     

Purification and characterization of extracellular pectate lyase from Bacillus subtilis

Bacillus subtilis strain SO113 secretes a pectate lyase which is produced during the exponential death phase of growth, just before sporulation. This extracellular pectate lyase, which produces unsaturated products from polygalacturonate, was purified 35-fold from the culture supernatant of Bacillus subtilis by a CM Sephadex chromatography. It has an isoelectric point of about 9.6 and an Mr of 42,000. Optimum activity occurred at pH 8.4 and at 42 degrees C. Calcium has a stimulative effect on the enzyme activity while EDTA leads to enzyme inactivation. The pectate lyase has a specific activity of 131 mumol of aldehyde groups per min and per mg of protein. The Km of the purified enzyme for polygalacturonic acid was 0.862 g.l-1 and the Vmax for polygalacturonic acid hydrolysis was 1.475 mumol of unsaturated products per min and per mg of protein. By using monoclonal antibodies raised against Erwinia chrysanthemi 3937 pectate lyases, it was shown that pectate lyases b and c of this strain are immunologically closely related to the Bacillus subtilis pectate lyase.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Microbial system for polysaccharide depolymerization: enzymatic route for xanthan depolymerization by Bacillus sp. strain GL1.

An enzymatic route for the depolymerization of a heteropolysaccharide (xanthan) in Bacillus sp. strain GL1, which was closely related to Brevibacillus thermoruber, was determined by analyzing the structures of xanthan depolymerization products. The bacterium produces extracellular xanthan lyase catalyzing the cleavage of the glycosidic bond between pyruvylated mannosyl and glucuronyl residues in xanthan side chains (W. Hashimoto et al., Appl. Environ. Microbiol. 64:3765-3768, 1998). The modified xanthan after the lyase reaction was then depolymerized by extracellular beta-D-glucanase to a tetrasaccharide, without the terminal mannosyl residue of the side chain in a pentasaccharide, a repeating unit of xanthan. The tetrasaccharide was taken into cells and converted to a trisaccharide (unsaturated glucuronyl-acetylated mannosyl-glucose) by beta-D-glucosidase. The trisaccharide was then converted to the unsaturated glucuronic acid and a disaccharide (mannosyl-glucose) by unsaturated glucuronyl hydrolase. Finally, the disaccharide was hydrolyzed to mannose and glucose by alpha-D-mannosidase. This is the first complete report on xanthan depolymerization by bacteria. Novel beta-D-glucanase, one of the five enzymes involved in the depolymerization route, was purified from the culture fluid. This enzyme was a homodimer with a subunit molecular mass of 173 kDa and was most active at pH 6.0 and 45 degrees C. The enzyme specifically acted on xanthan after treatment with xanthan lyase and released the tetrasaccharide.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species

Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetaiotaomicron. All three species produce two soluble cell-associated chondroitin lyases, chondroitin lyase I and II. Purified enzymes from the three species have similar pH optima, Km values, and molecular weights. However, peptide mapping experiments show that the chondroitin lyases from B. ovatus and Bacteroides sp. strain 3452A are not identical to those of B. thetaiotaomicron. A cloned gene that codes for the chondroitin lyase II from B. thetaiotaomicron hybridized on a Southern blot with DNA from B. ovatus or Bacteroides sp. strain 3452A only when low-stringency conditions were used. Antibody to chondroitin lyase II from B. thetaiotaomicron did not cross-react with chondroitin lyase II from B. ovatus or Bacteroides sp. strain 3452A. Chondroitin lyase activity in all three species was inducible by chondroitin sulfate. B. ovatus and Bacteroides sp. strain 3452A, like B. thetaiotaomicron, have outer membrane polypeptides that appear to be regulated by chondroitin sulfate, but the chondroitin sulfate-associated outer membrane polypeptides differ in molecular weight. Despite these differences, the ability of intact bacteria to utilize chondroitin sulfate, as indicated by growth yields in carbohydrate-limited continuous culture and the rate at which the chondroitin lyases were induced, was the same for all three species.     

A novel thermophilic pectate lyase containing two catalytic modules of Clostridium stercorarium

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca(2+) ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.     

Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers.     

Evidence that polygalacturonic acid may not be a major source of carbon and energy for some colonic Bacteroides species

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that polygalacturonic acid may not be a major source of carbon and energy for some colonic Bacteroides species.

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of pectin lyase and pectinase sequences

A total of 48 full-length protein sequences of pectin lyases from different source organisms available in NCBI were subjected to multiple sequence alignment, domain analysis, and phylogenetic tree construction. A phylogenetic tree constructed on the basis of the protein sequences revealed two distinct clusters representing pectin lyases from bacterial and fungal sources. Similarly, the multiple accessions of different source organisms representing bacterial and fungal pectin lyases also formed distinct clusters, showing sequence level homology. The sequence level similarities among different groups of pectinase enzymes, viz. pectin lyase, pectate lyase, polygalacturonase, and pectin esterase, were also analyzed by subjecting a single protein sequence from each group with common source organism to tree construction. Four distinct clusters representing different groups of pectinases with common source organisms were observed, indicating the existing sequence level similarity among them. Multiple sequence alignment of pectin lyase protein sequence of different source organisms along with pectinases with common source organisms revealed a conserved region, indicating homology at sequence level. A conserved domain Pec_Lyase_C was frequently observed in the protein sequences of pectin lyases and pectate lyases, while Glyco_hydro_28 domains and Pectate lyase-like β-helix clan domain are frequently observed in polygalacturonases and pectin esterases, respectively. The signature amino acid sequence of 41 amino acids, i.e. TYDNAGVLPITVN-SNKSLIGEGSKGVIKGKGLRIVSGAKNI, related with the Pec_Lyase_C is frequently observed in pectin lyase protein sequences and might be related with the structure and enzymatic function.     

A structural factor responsible for substrate recognition by Bacillus sp. GL1 xanthan lyase that acts specifically on pyruvated side chains of xanthan

Xanthan is a bacterial heteropolysaccharide composed of pentasaccharide repeating units, i.e., a cellobiose as a backbone and a trisaccharide consisting of two mannoses and one glucuronic acid as a side chain. Nonreducing terminal mannose residues of xanthan side chains are partially pyruvated. Bacillus sp. GL1 xanthan lyase, a member of polysaccharide lyase family 8, acts specifically on pyruvated side chains of xanthan and yields pyruvated mannose through a beta-elimination reaction by using a single Tyr255 residue as base and acid catalysts. Here we show structural factors for substrate recognition by xanthan lyase through X-ray crystallographic and mutational analyses. The enzyme accommodates mannose and pyruvated mannose at the -1 subsite, although both inhibitor and dissociation constants of the two monosaccharides indicated that the affinity of pyruvated mannose for xanthan lyase is much higher than that of mannose. The high affinity of pyruvated mannose is probably due to the formation of additional hydrogen bonds between the carboxyl group of pyruvated mannose and amino acid residues of Tyr315 and Arg612. Site-directed mutagenesis of the two residues demonstrated that Arg612 is a key residue in recognizing pyruvated mannose. Arg612 is located in the protruding loop covering the substrate, suggesting that the loop functions as a lid that is responsible for the proper accommodation of the substrate at the active site.     

Preparation of the methyl ester of hyaluronan and its enzymatic degradation

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1-->3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.     

Enzymatic properties of the highly thermophilic and alkaline pectate lyase Pel-4B from alkaliphilic Bacillus sp. strain P-4-N and the entire nucleotide and amino acid sequences

We cloned two genes for alkaline pectate lyase, pel-4A and pel-4B, from alkaline pectinase-producing alkaliphilic Bacillus sp. strain P-4-N. The pel-4B gene product Pel-4B was purified to homogeneity and characterized. The purified enzyme had an isoelectric point of pH 9.6 and a molecular mass of 35 kDa, values close to those of the pel-4A gene product Pel-4A. The pH and temperature optima for activity were as high as 11.5 and 70 degrees C, respectively, which are the highest among the pectate lyases reported to date. The mature Pel-4B (304 amino acids; 33,868 Da) was structurally related to the enzymes in the polysaccharide lyase family 1 and showed 35.6% identity with Pel-4A on the amino acid level. It showed significant homology to other pectate lyases in the same family, such as the enzymes from alkaliphilic Bacillus sp. strains KSM-P7 and KSM-P103 and the fungi Aspergillus nidulans and Colletotrichum gloeosporioides f. sp. malvae.     

Isolation of Mutant Alginate Lyases with Cleavage Specificity for Di-guluronic Acid Linkages

Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca²⁺, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.     

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T _m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.