Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

Abstract: β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity

Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.

Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling.

BackgroundGlycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions.ResultsSingle, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides.ConclusionsThe identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass

Determination of glycoside hydrolase specificities during hydrolysis of plant cell walls using glycome profiling.

BackgroundGlycoside hydrolases (GHs) are enzymes that hydrolyze polysaccharides into simple sugars. To better understand the specificity of enzyme hydrolysis within the complex matrix of polysaccharides found in the plant cell wall, we studied the reactions of individual enzymes using glycome profiling, where a comprehensive collection of cell wall glycan-directed monoclonal antibodies are used to detect polysaccharide epitopes remaining in the walls after enzyme treatment and quantitative nanostructure initiator mass spectrometry (oxime-NIMS) to determine soluble sugar products of their reactions.ResultsSingle, purified enzymes from the GH5_4, GH10, and GH11 families of glycoside hydrolases hydrolyzed hemicelluloses as evidenced by the loss of specific epitopes from the glycome profiles in enzyme-treated plant biomass. The glycome profiling data were further substantiated by oxime-NIMS, which identified hexose products from hydrolysis of cellulose, and pentose-only and mixed hexose-pentose products from the hydrolysis of hemicelluloses. The GH10 enzyme proved to be reactive with the broadest diversity of xylose-backbone polysaccharide epitopes, but was incapable of reacting with glucose-backbone polysaccharides. In contrast, the GH5 and GH11 enzymes studied here showed the ability to react with both glucose- and xylose-backbone polysaccharides.ConclusionsThe identification of enzyme specificity for a wide diversity of polysaccharide structures provided by glycome profiling, and the correlated identification of soluble oligosaccharide hydrolysis products provided by oxime-NIMS, offers a unique combination to understand the hydrolytic capabilities and constraints of individual enzymes as they interact with plant biomass

End products from exhaustive hydrolysis of locust bean gum by SACTE_2347 determined by HPLC.

<p>The three major products identified by comparison of elution times with purified commercial standards were ¹G,²G-M3 (<b>8</b>), ¹G-M2 (<b>5</b>), and M2 (<b>2</b>).</p

Schematic diagram of the binding subsites of SACTE_2347 correlated with reaction of purified oligomannosides and galactosyl-substituted oligomannosides.

<p>The active site schematic shows the positions of sugar binding subsites, the catalytic residues Glu178 and Glu272, and the position of loops L1 and L2. Mannosyl groups (grey circles) and galactosyl groups (black circles) of purified substrates studies are aligned in the −3 to +2 subsites under the schematic of the active site channel. Loop L1 blocks binding of a substituted mannosyl group in either the +1 of +2 subsites. The space between L1 and L2 allows placement of a substituted mannosyl group in the −1 subsite, while shortened L2 allows placement of a substituted mannosyl group into the −2 subsite. All reaction products can be rationalized to arise from hydrolysis of the glycosidic bond between the −1 and +1 subsites after accounting for steric interactions with L1 and L2.</p

Kinetic constants determined for SACTE_2347 variants.

a<p>Pure β-1,4 d-mannan.</p>b<p>Acetylated glucomannan contain mannan (60%) and glucose (40%).</p>c<p>Locust bean gum is a natural galactomannan with composition of ∼3.5 mannose per galactose.</p>d<p>IL-pine has the following composition: 34% glucose; 9% xylose; 8% mannose; 4% arabinose, and 8% galactose.</p><p>SACTE_2347 did not hydrolyze cellulose, xylan and other polysaccharides described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#s4" target="_blank">Materials and Methods</a>, and likewise did not react with fluorogenic small molecule analogs.</p

Atomic resolution structure of SACTE_2347.

<p>A, 2Fo-Fc electron density map of eight conserved residues in the GH5 family, contoured at 1.3 σ. Hydrogen atoms were included in the refinement of the high-resolution data. B, Comparison of the active site channels of SACTE_2347 (green) and TfManA (blue). Residues that form the surface of the channel are highlighted in gray, and the positions of loops L1 and L2 are indicated. Positions of the catalytic residues (Glu178 and Glu273 in SACTE_2347_34kDa) are shown in red. Mannobiose observed in the −3 and −2 subsites of the TfManA structure is shown as ball and sticks.</p

Peptide sequences identified by mass spectrometry.

a<p>The full protein sequence of SACTE_2347, annotated with the positions of these peptides is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone.0094166.s002" target="_blank">Figure S2</a>. The names of peptides are also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone-0094166-g002" target="_blank">Figure 2</a>.</p>b<p>Observed <i>m/z</i>.</p

Phylogeny of GH5 subfamily 8 including SACTE_2347.

<p>The phylogenic tree was constructed from all sequences assigned to the GH5 subfamily 8. Names shown are GenBank accession codes. Black circles indicate enzymes that have been experimentally verified to exhibit β-mannanase activity; black stars indicate three enzymes whose structures have been determined besides SACTE_2347. These are: <i>Thermomonospora fusca</i>, AAZ54938.1, PDB 1BQC, 2MAN, 3MAN; <i>Bacillus</i> sp. N16-5, AAT06599.1, PDB 2WHJ, 2WJL, 3JUG; <i>Bacillus</i> sp. JAMB-602, BAD99527.1, PDB 1WKY.</p