Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZPss.

HrpZ-a harpin from Pseudomonas syringae-is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities

Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues

Synthesis of long-chain chitooligosaccharides by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568

We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40°C, where it showed a Km of 83 mg ml−1, a kcat of 3.9 × 102 h−1, and a kcat/Km of 4.7 h mg−1 ml−1 on colloidal chitin. On chitobiose, the Km, kcat, and kcat/Km were 203 μM, 1.3 × 102 h−1, and 0.62 h−1 μM−1, respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase

Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases

Glycosyl hydrolase (GH) family 18 chitinases (Chi) and family 33 chitin binding proteins (CBPs) from Bacillus thuringiensis serovar kurstaki (BtChi and BtCBP), B. licheniformis DSM13 (BliChi and BliCBP) and Serratia proteamaculans 568 (SpChiB and SpCBP21) were used to study the efficiency and synergistic action of BtChi, BliChi and SpChiB individually with BtCBP, BliCBP or SpCBP21. Chitinase assay revealed that only BtChi and SpChiB showed synergism in hydrolysis of chitin, while there was no increase in products generated by BliChi, in the presence of the three above mentioned CBPs. This suggests that some (specific) CBPs are able to exert a synergistic effect on (specific) chitinases. A mutant of BliChi, designated as BliGH, was constructed by deleting the C-terminal fibronectin III (FnIII) and carbohydrate binding module 5 (CBM5) to assess the contribution of FnIII and CBM5 domains in the synergistic interactions of GH18 chitinases with CBPs. Chitinase assay with BliGH revealed that the accessory domains play a major role in making BliChi an efficient enzyme. We studied binding of BtCBP and BliCBP to α- andβ-chitin. The BtCBP, BliCBP or SpCBP21 did not act synergistically with chitinases in hydrolysis of the chitin, interspersed with other polymers, present in fungal cell walls

Transglycosylation by chitinase D from Serratia proteamaculans improved through altered substrate interactions

We describe improvement of the transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced less proportion of TG products up to 90 min, with 2 mM chitotetraose as the substrate and hydrolysed the products subsequently. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. Rest of the three mutants, M226A, Y228A and R284A displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S and S116G, at the catalytic groove increased TG activity, while W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of W247 at the solvent accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared to SpChiD, respectively. The double mutant E159D/F64W regained the activity with accumulation of 6.0% of chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of quantity of TG products produced and the extended duration of TG activity

Multiple chitinases of an endophytic Serratia proteamaculans 568 generate chitin oligomers

Serratia proteamaculans 568 genome revealed the presence of four family 18 chitinases (Sp ChiA, Sp ChiB, Sp ChiC, and Sp ChiD). Heterologous expression and characterization of Sp ChiA, Sp ChiB, and Sp ChiC showed that these enzymes were optimally active at pH 6.0–7.0, and 40 °C. The three Sp chitinases displayed highest activity/binding to β-chitin and showed broad range of substrate specificities, and released dimer as major end product from oligomeric and polymeric substrates. Longer incubation was required for hydrolysis of trimer for the three Sp chitinases. The three Sp chitinases released up to tetramers from colloidal chitin substrate. Sp ChiA and Sp ChiB were processive chitinases, while Sp ChiC was a non-processive chitinase. Based on the known structures of ChiA and ChiB from S. marcescens, 3D models of Sp ChiA and Sp ChiB were generated

Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases of plants

Fungal diseases of plants continue to contribute to heavy crop losses in spite of the best control efforts of plant pathologists. Breeding for disease-resistant varieties and the application of synthetic chemical fungicides are the most widely accepted approaches in plant disease management. An alternative approach to avoid the undesired effects of chemical control could be biological control using antifungal bacteria that exhibit a direct action against fungal pathogens. Several biocontrol agents, with specific fungal targets, have been registered and released in the commercial market with different fungal pathogens as targets. However, these have not yet achieved their full commercial potential due to the inherent limitations in the use of living organisms, such as relatively short shelf life of the products and inconsistent performance in the field. Different mechanisms of action have been identified in microbial biocontrol of fungal plant diseases including competition for space or nutrients, production of antifungal metabolites, and secretion of hydrolytic enzymes such as chitinases and glucanases. This review focuses on the bacterial chitinases that hydrolyze the chitinous fungal cell wall, which is the most important targeted structural component of fungal pathogens. The application of the hydrolytic enzyme preparations, devoid of live bacteria, could be more efficacious in fungal control strategies. This approach, however, is still in its infancy, due to prohibitive production costs. Here, we critically examine available sources of bacterial chitinases and the approaches to improve enzymatic properties using biotechnological tools. We project that the combination of microbial and recombinant DNA technologies will yield more effective environment-friendly products of bacterial chitinases to control fungal diseases of crops

Sequence alignment and domain organisation for <i>Sp</i> CBPs.

<p>(A) Full-length sequences of <i>Sp</i> CBP21, <i>Sp</i> CBP28, <i>Sp</i> CBP50 and <i>Sm</i> CBP21 (CBP21 from <i>S. marcescens</i>) were aligned using clustalw2. Residues that are thought to be located in the binding surface for chitin present in <i>Sm</i> CBP21, <i>Sp</i> CBP21, <i>Sp</i> CBP50 and not present in <i>Sp</i> CBP28 are shaded in yellow (as derived from the crystal structure of <i>Sm</i> CBP21, as well as mutagenesis studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036714#pone.0036714-VaajeKolstad2" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036714#pone.0036714-VaajeKolstad3" target="_blank">[8]</a>). Residue involved in the chitin-binding and functional properties of <i>Sm</i> CBP21 but also conserved in <i>Sp</i> CBP28 are shaded grey. The arrow indicates the terminal amino acid of the N-terminal signal sequence for respective CBPs. (B) The sequences of <i>Sp</i> CBPs were submitted to SMART domain data base (<a href="http://smart.embl-heidelberg.de/" target="_blank">http://smart.embl-heidelberg.de/</a>). The part indicated in red colour shows the signal peptide and the region Chitin_bind_3 indicates the chitin binding domain.</p

List of primers used for the amplification of genes coding for chitin binding proteins and chitinases from <i>Serratia proteamaculans</i> 568.

a<p>Sequences underlined represent restriction sites.</p

β-chitin hydrolysis enhancing effects of <i>Sp</i> CBP21 and <i>Sp</i> CBP50 with <i>Sp</i> ChiD.

<p>Reaction mixture (1 mL) containing 0.25 mg/mL of β-chitin, 0.25 µM/0.50 µM/0.75 µM/1.0 µM <i>Sp</i> ChiD incubated individually with 0.3 µM of <i>Sp</i> CBP21/<i>Sp</i> CBP50 or combining both <i>Sp</i> CBP21 and <i>Sp</i> CBP50 (0.15 µM +0.15 µM/0.30 µM +0.30 µM), in 50 mM sodium phosphate buffer pH 7.0. After incubation at 37°C for 24 h at 1000 rpm, 100 µL of reaction mixture was transferred. To this 100 µL of 0.02N NaOH was added to stop the reaction and stored at −20°C until products quantification by standard reducing end assay. Vertical bars represent standard deviation of triplicate experiments.</p