Biochemical and molecular characterization of poly(aspartic acid) hydrolase-2 from sphingomonas sp. KT-1

Poly(aspartic acid) (PAA) hydrolase-2 was purified from crude soluble cellular extracts of Sphingomonas sp. KT-1 (JCM10459) and characterized to elucidate the mechanism of alpha,beta-poly(d,l-aspartic acid) (tPAA) biodegradation. The molecular mass of PAA hydrolase-2 was 42 kDa, and the isoelectric point was 9.6. The optimum values of pH and temperature for the hydrolysis of alpha-di(l-aspartic acid) by PAA hydrolase-2 were 7.0 and 55 degrees C, respectively. The effect of inhibitors on the hydrolysis of alpha-di(l-aspartic acid) showed that the activity of PAA hydrolase-2 was significantly inhibited by EDTA. Thermally synthesized tPAA was hydrolyzed in the presence of two enzymes, PAA hydrolase-1 and PAA hydrolase-2, to generate aspartic acid. The PAA hydrolase-2 was capable of hydrolyzing alpha-poly(l-aspartic acid) of high molecular weights but had limited activity for tPAA. These results lead us to propose the following mechanism. First, PAA hydrolase-1 hydrolyzes tPAA to yield oligo(aspartic acid) via an endo-mode cleavage, and subsequently, PAA hydrolase-2 hydrolyzes the resultant oligo(aspartic acid) to yield aspartic acid. Analysis of hydrolyzed products from alpha- and beta-penta(l-aspartic acid) revealed that PAA hydrolase-2 catalyzed the exo-mode hydrolysis of alpha- and beta-penta (l-aspartic acid). The gene encoding PAA hydrolase-2 from Sphingomonas sp. KT-1 was cloned, and genetic analysis showed that the deduced amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to the M20/M25/M40 family of proteins, from Caulobacter crescentus CB15.     

Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1

We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.     

聚天冬氨酸功能高分子材料研究进展

首先概述了聚天冬氨酸广泛用于肥料增效剂等传统领域的最新进展,然后对聚天冬功能材料应用研究进行了综述,其中包括生物医学影像材料、智能凝胶材料、药物缓释材料、绿色分散剂以及燃料吸水剂等,最后对聚天冬氨酸功能材料未来的研究方向进行了评述。     

Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.     

Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1

A DNA fragment carrying the gene encoding poly(3-hydroxybutyrate) (P(3HB)) depolymerase was cloned from the genomic DNA of Marinobacter sp. DNA sequencing analysis revealed that the Marinobacter sp. P(3HB) depolymerase gene is composed of 1734 bp and encodes 578 amino acids with a molecular mass of 61,757 Da. A sequence homology search showed that the deduced protein contains the signal peptide, catalytic domain (CD), cadherin-type linker domain (LD), and two substrate-binding domain (SBD). The fusion proteins of glutathione S-transferase (GST) with the CD showed the hydrolytic activity for denatured P(3HB) (dP(3HB)), P(3HB) emulsion (eP(3HB)) and p-nitrophenylbutyrate. On the other hand, the fusion proteins lacking the SBD showed much lower hydrolytic activity for dP(3HB) compared to the proteins containing both CD and SBD. In addition, binding tests revealed that the SBDs are specifically bound not to eP(3HB) but dP(3HB). These suggest that the SBDs play a crucial role in the enzymatic hydrolysis of dP(3HB) that is a solid substrate.     

Nmr study of poly(aspartic acid). II. α- and β-Peptide bonds in poly(aspartic acid) prepared by common methods

The nature of peptide bonds in poly(aspartic acid) prepared by debenzylation of poly(β-benzyl-L -aspartate) under various conditions has been studied by means of nmr spectroscopy. It was established that the majority of the polymers prepared, as well as the commercially obtained polymer, contained aspartic acid linked in both α- and β-peptide bonds. The purest polymer, having practically undetectable amounts of β-bond, was prepared by debenzylation by HBr in trifluoroacetic acid. It was established that the β-bonds are formed via succinimides.     

Nmr study of poly(aspartic acid). I. α- and β-Peptide bonds in poly(aspartic acid) prepared by thermal polycondensation

The structure of poly(aspartic acid) prepared by thermal polycondensation has been studied by means of nmr spectroscopy. The analysis of the ¹³C-nmr spectra of the polymer at various pH values and comparison with the spectrum of poly(α-L -aspartic acid) revealed that the polymer contained aspartic acid linked in α- and β-peptide bonds. The mole fraction of the β-peptide bonds has been found to be 0.8 ± 0.1. The significance of the results for the evolutionary theory of S. W. Fox is mentioned.     

Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1

Poly(L-lactic acid) (PLA)-degrading Amycolatopsis sp. strains K104-1 and K104-2 were isolated by screening 300 soil samples for the ability to form clear zones on the PLA-emulsified mineral agar plates. Both of the strains assimilated >90% of emulsified 0.1% (wt/vol) PLA within 8 days under aerobic conditions. A novel PLA depolymerase with a molecular weight of 24,000 was purified to homogeneity from the culture supernatant of strain K104-1. The purified enzyme degraded high-molecular-weight PLA in emulsion and in solid film, ultimately forming lactic acid. The optimum pH for the enzyme activity was 9.5, and the optimum temperature was 55 to 60 degrees C. The PLA depolymerase also degraded casein and fibrin but did not hydrolyze collagen type I, triolein, tributyrin, poly(beta-hydroxybutyrate), or poly(epsilon-caprolactone). The PLA-degrading and caseinolytic activities of the enzyme were inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but were not significantly affected by soybean trypsin inhibitor, N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethyl ketone, and Streptomyces subtilisin inhibitor. Thus, Amycolatopsis sp. strain K104-1 excretes the unique PLA-degrading and fibrinolytic serine enzyme, utilizing extracellular polylactide as a sole carbon source.     

Overexpression, purification, and characterization of ATP-NAD kinase of Sphingomonas sp. A1

The NAD kinase gene (nadK) of Sphingomonas sp. A1 was cloned and then overexpressed in Escherichia coli, and the gene product (NadK) was purified from the E. coli cells through five steps with a 25% yield of activity. NadK was a homodimer of 32 kDa subunits, utilized ATP or other nucleoside triphosphates, but not inorganic polyphosphates, as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 8.0 and 50-55 degrees C, and was designated as ATP-NAD kinase (NadK). NadK showed no NADH kinase activity and was slightly inhibited by NADP(H). Precursors for NAD biosynthesis such as quinolinic acid, nicotinic acid mononucleotide, nicotinic acid adenine dinucleotide, and nicotinic acid had no effect on the NadK activity, as observed in the cases of the NAD kinases of Micrococcus flavus, Mycobacterium tuberculosis, and E. coli. Taken together with the report that the NAD kinase of Bacillus subtilis is activated by quinolinic acid [J. Bacteriol. 185 (2003) 4844], it is indicated that the regulatory patterns of NAD kinases differ even among bacterial NAD kinases.     

Genetic analysis of phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH

Phenoxyalkanoic acid degradation is well studied in Beta- and Gammaproteobacteria, but the genetic background has not been elucidated so far in Alphaproteobacteria. We report the isolation of several genes involved in dichlor- and mecoprop degradation from the alphaproteobacterium Sphingomonas herbicidovorans MH and propose that the degradation proceeds analogously to that previously reported for 2,4-dichlorophenoxyacetic acid (2,4-D). Two genes for alpha-ketoglutarate-dependent dioxygenases, sdpA(MH) and rdpA(MH), were found, both of which were adjacent to sequences with potential insertion elements. Furthermore, a gene for a dichlorophenol hydroxylase (tfdB), a putative regulatory gene (cadR), two genes for dichlorocatechol 1,2-dioxygenases (dccA(I/II)), two for dienelactone hydrolases (dccD(I/II)), part of a gene for maleylacetate reductase (dccE), and one gene for a potential phenoxyalkanoic acid permease were isolated. In contrast to other 2,4-D degraders, the sdp, rdp, and dcc genes were scattered over the genome and their expression was not tightly regulated. No coherent pattern was derived on the possible origin of the sdp, rdp, and dcc pathway genes. rdpA(MH) was 99% identical to rdpA(MC1), an (R)-dichlorprop/alpha-ketoglutarate dioxygenase from Delftia acidovorans MC1, which is evidence for a recent gene exchange between Alpha- and Betaproteobacteria. Conversely, DccA(I) and DccA(II) did not group within the known chlorocatechol 1,2-dioxygenases, but formed a separate branch in clustering analysis. This suggests a different reservoir and reduced transfer for the genes of the modified ortho-cleavage pathway in Alphaproteobacteria compared with the ones in Beta- and Gammaproteobacteria.     

Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.     

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (K_m) and specificity constants (K_cat/K_m) of the enzyme were determined to be 15 μM and 511 s⁻¹ M⁻¹ × 10⁴ for gentisate and 754 μM and 20 s⁻¹ M⁻¹ × 10⁴ for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.Large amounts of aromatic compounds have been released into the environment during the last decades as a result of extensive production of industrial chemicals and agricultural applications of pesticides. Many of these compounds, particularly the chlorinated derivatives, are toxic, even at low concentrations, and persist in the environment (14, 39). Numerous microorganisms have been isolated which degrade xenobiotic aromatic compounds through aerobic or anaerobic degradative reactions (16, 17, 34, 46, 47). A wide variety of polycyclic and homocyclic aromatic compounds are aerobically transformed to a limited number of central dihydroxylated intermediates like catechol, protocatechuate, or gentisate. Whereas catabolic pathways for catechol and protocatechuate have been extensively characterized (22, 35), little is known about gentisate degradation.Gentisic acid (2,5-dihydroxybenzoic acid) is a key intermediate in the aerobic degradation of such aromatic compounds as dibenzofuran (15), naphthalene (18, 48), salicylate (40, 55), anthranilate (32), and 3-hydroxybenzoate (26). Degradation of gentisate is initiated by gentisate 1,2-dioxygenase (GDO; EC 1.13.11.4, gentisate:oxygen oxidoreductase), which cleaves the aromatic ring between the carboxyl and the vicinal hydroxyl group to form maleylpyruvate (30). Maleylpyruvate can be converted to central metabolites of the Krebs cycle either by cleavage to pyruvate and maleate (5, 24) or by isomerization to fumarylpyruvate and subsequent cleavage to fumarate and pyruvate (10, 31, 51).Of the two well-studied classes of ring cleavage dioxygenases, intradiol enzymes, such as catechol 1,2-dioxygenase or protocatechuate 3,4-dioxygenase, contain an Fe³⁺ atom in the catalytic center and cleave the aromatic substrate between two vicinal hydroxyl groups (7, 37, 38), whereas dioxygenases of the extradiol class, such as catechol 2,3-dioxygenase or protocatechuate 4,5-dioxygenase, contain Fe²⁺ and cleave the aromatic substrate adjacent to two vicinal hydroxyl groups (1, 13). Gentisate 1,2-dioxygenase cleaves aromatic rings containing hydroxyl groups situated para to one another. Although the mechanism of oxygen activation was proposed to be similar to that of enzymes of the extradiol dioxygenase class (20), and the active center contains Fe²⁺ (11, 21, 29, 49, 51), the Fe²⁺ is not bound to the enzyme by electron-donating ligands such as cysteine or tyrosine (21) as is the case for extradiol-cleaving dioxygenases (19). It is being assumed, therefore, that GDO represents a novel class of ring-cleaving dioxygenases.GDOs have been purified and characterized from gram-positive bacteria of the genera Bacillus and Rhodococcus (29, 50) and gram-negative bacteria of the genera Klebsiella, Comamonas, and Moraxella (11, 21, 49), and amino-terminal sequences of GDOs from Comamonas testosteroni and Comamonas acidovorans have been determined (21), but until now, no complete sequence of any GDO or of a gene encoding GDO has been reported. Here we describe the cloning and sequencing of the gene encoding the GDO from Sphingomonas sp. strain RW5 and its partial characterization. This GDO represents a novel class of dioxygenases with very low similarity to any other known ring-cleaving dioxygenases.     

Formation of core-shell type biodegradable polymeric micelles from amphiphilic poly(aspartic acid)-block-polylactide diblock copolymer

Poly(aspartic acid)-block-polylactide diblock copolymers (PAsp-b-PLAs) having both hydrophilic and hydrophobic segments of various lengths were synthesized. These PAsp-b-PLA diblock copolymers formed polymeric micelles consisting of a hydrophobic PLA core and a hydrophilic, pH-sensitive PAsp shell in aqueous solution. The effects of the segment length of both the PLA and the PAsp portions and the pH of the solution on the shapes and sizes of the PAsp-b-PLA polymeric micelles were investigated. The results indicated a balance between the effects of electrostatic repulsion, hydrogen bonding in the PAsp shell layer, and hydrophobic interactions in the PLA core determine the sizes of the PAsp-b-PLA polymeric micelles. Moreover, the PAsp-b-PLA polymeric micelles did not possess any cytotoxic activity against L929 fibroblast cells. The obtained polymeric micelle should be useful for biodegradable biomedical materials such as drug delivery vehicle.     

Purification and Characterization of a Three-Component Salicylate 1-Hydroxylase from Sphingomonas sp. Strain CHY-1

In the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs), salicylate hydroxylases catalyze essential reactions at the junction between the so-called upper and lower catabolic pathways. Unlike the salicylate 1-hydroxylase from pseudomonads, which is a well-characterized flavoprotein, the enzyme found in sphingomonads appears to be a three-component Fe-S protein complex, which so far has not been characterized. Here, the salicylate 1-hydroxylase from Sphingomonas sp. strain CHY-1 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component, designated PhnII, exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. In the presence of purified reductase (PhnA4) and ferredoxin (PhnA3) components, PhnII catalyzed the hydroxylation of salicylate to catechol with a maximal specific activity of 0.89 U/mg and showed an apparent K_m for salicylate of 1.1 ± 0.2 μM. The hydroxylase exhibited similar activity levels with methylsalicylates and low activity with salicylate analogues bearing additional hydroxyl or electron-withdrawing substituents. PhnII converted anthranilate to 2-aminophenol and exhibited a relatively low affinity for this substrate (K_m, 28 ± 6 μM). 1-Hydroxy-2-naphthoate, which is an intermediate in phenanthrene degradation, was not hydroxylated by PhnII, but it induced a high rate of uncoupled oxidation of NADH. It also exerted strong competitive inhibition of salicylate hydroxylation, with a K_i of 0.68 μM. The properties of this three-component hydroxylase are compared with those of analogous bacterial hydroxylases and are discussed in light of our current knowledge of PAH degradation by sphingomonads.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Isolation and characterization of polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.     

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.     

Substrate recognition by family 7 alginate lyase from Sphingomonas sp. A1

Sphingomonas sp. A1 alginate lyase A1-II′, a member of polysaccharide lyase family 7, shows a broad substrate specificity acting on poly α-L-guluronate (poly(G)), poly β-D-mannuronate (poly(M)) and the heteropolymer (poly(MG)) in alginate molecules. A1-II′ with a glove-like β-sandwich as a basic scaffold forms a cleft covered with two lid loops (L1 and L2). Here, we demonstrate the loop flexibility for substrate binding and structural determinants for broad substrate recognition and catalytic reaction. The two loops associate mutually over the cleft through the formation of a hydrogen bond between their edges (Asn141 and Asn199). A double mutant, A1-II′ N141C/N199C, has a disulfide bond between Cys141 and Cys199, and shows little enzyme activity. Adding dithiothreitol to the enzyme reaction mixture leads to a tenfold increase in its molecular activity, suggesting the significance of flexibility in lid loops for accommodating the substrate into the active cleft. In alginate trisaccharide (GGG or MMG)-bound A1-II′ Y284F, the enzyme interacts appropriately with substrate hydroxyl groups at subsites + 1 and + 2 and accommodates G or M, while substrate carboxyl groups are strictly recognized by specific residues. This mechanism for substrate recognition enables A1-II′ to show the broad substrate specificity. The structure of A1-II′ H191N/Y284F complexed with a tetrasaccharide bound at subsites − 1 to + 3 suggests that Gln189 functions as a neutralizer for the substrate carboxyl group, His191 as a general base, and Tyr284 as a general acid. This is, to our knowledge, the first report on the structure and function relationship in family 7.     

Metabolism of dibenzo-p-dioxin by Sphingomonas sp. strain RW1.

In the course of our screening for dibenzo-p-dioxin-utilizing bacteria, a Sphingomonas sp. strain was isolated from enrichment cultures inoculated with water samples from the river Elbe. The isolate grew with both the biaryl ethers dibenzo-p-dioxin and dibenzofuran (DF) as the sole sources of carbon and energy, showing doubling times of about 8 and 5 h, respectively. Biodegradation of the two aromatic compounds initially proceeded after an oxygenolytic attack at the angular position adjacent to the ether bridge, producing 2,2',3-trihydroxydiphenyl ether or 2,2',3-trihydroxybiphenyl from the initially formed dihydrodiols, which represent extremely unstable hemiacetals. Results obtained from determinations of enzyme activities and oxygen consumption suggest meta cleavage of the trihydroxy compounds. During dibenzofuran degradation, hydrolysis of 2-hydroxy-6-oxo-6-(2-hydroxyphenyl)-hexa-2,4-dienoate yielded salicylate, which was branched into the catechol meta cleavage pathway and the gentisate pathway. Catechol obtained from the product of meta ring fission of 2,2',3-trihydroxydiphenyl ether was both ortho and meta cleaved by Sphingomonas sp. strain RW1 when this organism was grown with dibenzo-p-dioxin.