Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that has a 38-kb gene cluster for the utilization of arabinan, a branched polysaccharide that is part of the plant cell wall. The bacterium encodes a unique three-component regulatory system (araPST) that includes a sugar-binding lipoprotein (AraP), a histidine sensor kinase (AraS), and a response regulator (AraT) and lies adjacent to an ATP-binding cassette (ABC) arabinose transport system (araEGH). The lipoprotein (AraP) specifically bound arabinose, and gel mobility shift experiments showed that the response regulator, AraT, binds to a 139-bp fragment corresponding to the araE promoter region. Taken together, the results showed that the araPST system appeared to sense extracellular arabinose and to activate a specific ABC transporter for arabinose (AraEGH). The promoter regions of the arabinan utilization genes contain a 14-bp inverted repeat motif resembling an operator site for the arabinose repressor, AraR. AraR was found to bind specifically to these sequences, and binding was efficiently prevented in the presence of arabinose, suggesting that arabinose is the molecular inducer of the arabinan utilization system. The expression of the arabinan utilization genes was reduced in the presence of glucose, indicating that regulation is also mediated via a catabolic repression mechanism. The cluster also encodes a second putative ABC sugar transporter (AbnEFJ) whose sugar-binding lipoprotein (AbnE) was shown to interact specifically with linear and branched arabino-oligosaccharides. The final degradation of the arabino-oligosaccharides is likely carried out by intracellular enzymes, including two ␣-L-arabinofuranosidases (AbfA and AbfB), a -L-arabinopyranosidase (Abp), and an arabinanase (AbnB), all of which are encoded in the 38-kb cluster.The natural degradation of biomass from plants is a key step in the carbon cycle (53,69,79). This process is carried out mainly by microorganisms that can be found either free or as part of the digestive system in higher animals (76). The three main polysaccharides in the plant cell wall are cellulose, hemicellulose, and pectin, which are rigidified by lignin, a heterogeneous aromatic polymer (28, 60). Pectin is a complex polysaccharide and may account for up to 30% of the dry weight of the plant cell wall (46). Arabinan is a pectic polysaccharide consisting of a backbone of ␣-1,5-linked L-arabinofuranosyl units, which are further decorated mainly with ␣-1,2-and ␣-1,3-linked arabinofuranosides (46).Three general strategies are taken by the microbial world for plant cell wall degradation and can be described as follows. Anaerobic bacteria, such as Clostridium spp., have evolved unique multienzyme complexes, named cellulosomes, that integrate many cellulolytic and hemicellulolytic enzymes and mediate both the attachment of the cell to the crystalline polymer and its controlled hydrolysis (9,16,23,65). Aerobic fungi, such as Trichoderma and Aspergillus, secrete a large variety of free cellulases, hemicellulases...
Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His 6 -XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the ؊53 and the ؉34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 M XynC. The His 6 -tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (⌬C p ) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system.
A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK,kdgA, uxaC, uxuA, anduxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.
a-d-Glucuronidases cleave the a-1,2-glycosidic bond of the 4-O-methyl-d-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The a-dglucuronidase from Bacillus stearothermophilus T-6 was overexpressed in Escherichia coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture. Based on gel filtration, the native protein is comprised of two identical subunits. Kinetic constants with aldotetraouronic acid as a substrate, at 55 8C, were a K m of 0.2 mm, and a specific activity of 42 U´mg 21 (k cat 54.9 s 21 ). The enzyme was most active at 65 8C, pH 5.5± 6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 8C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 8C. Truncated forms of the enzyme, lacking either 126 amino acids from its N-terminus or 81 amino acids from its C-terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein. To identify the potential catalytic residues, sitedirected mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu3923Cys and Asp3643Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.
␣-Glucuronidases cleave the ␣-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the ␣-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the ␣-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-D-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp 364 and Glu 392 , activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu 285 cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu 285 is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.
Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 A resolution to a final R factor of 15.0% and an R(free) of 19.0%. As expected, the structure forms the classical (alpha/beta)(8) fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.
The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase is metal-dependent in one class of organisms and metal-independent in another. We have used a rapid transient kinetic approach combined with site-directed mutagenesis to characterize the role of the metal ion as well as to explore the catalytic mechanisms of the two classes of enzymes. In the metal-dependent Aquifex pyrophilus KDO8P synthase, Cys 11 was replaced by Asn (ApC11N), and in the metal-independent Escherichia coli KDO8P synthase a reciprocal mutation, Asn 26 to Cys, was prepared (EcN26C). The ApC11N mutant retained about 10% of the wild-type maximal activity in the absence of metal ions. Addition of divalent metal ions did not affect the catalytic activity of the mutant enzyme and its catalytic efficiency (k cat /K m ) was reduced by only ϳ12-fold, implying that the ApC11N KDO8P synthase mutant has become a bone fide metal-independent enzyme. The isolated EcN26C mutant had similar metal content and spectral properties as the metal-dependent wild-type A. pyrophilus KDO8P synthase. EDTA-treated EcN26C retained about 6% of the wild-type activity, and the addition of Mn 2؉ or Cd 2؉ stimulated its activity to ϳ30% of the wild-type maximal activity. This suggests that EcN26C KDO8P synthase mutant has properties similar to that of metal-dependent KDO8P synthases. The combined data indicate that the metal ion is not directly involved in the chemistry of the KDO8P synthase catalyzed reaction, but has an important structural role in metal-dependent enzymes in maintaining the correct orientation of the substrates and/or reaction intermediate(s) in the enzyme active site.The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) 1 synthase (EC 4.1.2.16) catalyzes the condensation reaction between D-arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P and inorganic phosphate (P i ) (see Scheme 1) (1). This enzymatic reaction plays an essential role in the assembly process of lipopolysaccharides of most Gram-negative bacteria, and therefore represents an attractive target for the design of novel antibacterial drugs (2, 3). While earlier studies on Escherichia coli KDO8P synthase have established that this enzyme does not require metals (1), it has recently been demonstrated that enzymes from the hyperthermophilic bacteria Aquifex aeolicus (4), Aquifex pyrophilus (5), and from the pathogenic bacterium Helicobacter pylori (6), require a divalent metal cofactor for catalysis. Furthermore, phylogenetic analysis (7, 8) suggests that KDO8P synthases from other pathogenic bacteria, i.e. Chlamydia trachomatis, Chlamydia pneumoniae, and Campylobacter jejuni, may also be metal-dependent enzymes. This is reminiscent of another class of enzymes, aldolases, which catalyze a similar aldol-type C-C bond formation (9). Class I aldolases, which are primarily found in animals and higher plants, do not require a metal cofactor for catalysis. In contrast, Class II aldolases, found predominantly in prokaryotes, use a divalent metal cofactor that...
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