Recombinant Oryza sativa subsp. japonica Beta-glucosidase 12 (BGLU12)

What are the critical catalytic residues in Os4BGlu12?

Os4BGlu12 contains highly conserved catalytic residues characteristic of GH1 family enzymes. The catalytic nucleophile has been identified as Glu393, which is involved in the formation of the glycosyl-enzyme intermediate during catalysis. The position of this residue in the G2F (2-deoxy-2-fluoroglucose) structure was found to be more similar to that of Sinapsis alba myrosinase G2F complex than to other O-glucosidases . Site-directed mutagenesis studies of homologous enzymes indicate that mutation of the conserved catalytic glutamic acid residues completely abolishes β-glucosidase activity .

What substrates does Os4BGlu12 hydrolyze?

Os4BGlu12 demonstrates versatile substrate specificity. It can hydrolyze:

• β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues

• β-(1,3)-linked disaccharide laminaribiose

• Various glycosides

Additionally, Os4BGlu12 exhibits significant thioglucosidase activity, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides compared to the O-glucosides .

How can recombinant Os4BGlu12 be expressed in bacterial systems?

Os4BGlu12 has been successfully expressed as a fusion protein with an N-terminal thioredoxin/His6 tag in Escherichia coli strain Origami B (DE3). This expression system yields soluble protein with appropriate folding for enzymatic activity . The protocol involves:

• Cloning the Os4bglu12 cDNA into an appropriate expression vector

• Transforming E. coli Origami B (DE3) cells with the construct

• Inducing expression under optimized conditions

• Harvesting cells and extracting the soluble protein fraction

This method produces approximately 6 mg of purified protein per liter of LB medium .

What purification strategies are effective for recombinant Os4BGlu12?

Purification of recombinant Os4BGlu12 typically involves a multi-step process:

• Initial capture using immobilized metal affinity chromatography (IMAC) to bind the His6 tag

• Tag removal through enzymatic cleavage

• Secondary purification steps to remove the cleaved tag and other contaminants

• Final polishing steps to achieve high purity (~95%)

This purification workflow yields protein suitable for crystallization and enzymatic studies. The purified protein can be subsequently used for various analyses, including activity assays and crystallization trials .

What conditions are optimal for crystallizing Os4BGlu12?

Native Os4BGlu12 crystals have been successfully obtained under the following conditions:

• 19%(w/v) PEG 3350

• 0.1 M Tris–HCl pH 8.5

• 0.16 M NaCl

• Hanging-drop vapor diffusion method with microseeding

• Temperature: 288 K (15°C)

For complexes with inhibitors such as 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside (DNP2FG), slightly modified conditions have been used:

• 19%(w/v) PEG 2000

• 0.1 M Tris–HCl pH 8.5

• 0.16 M NaCl

What diffraction data collection parameters have been used for Os4BGlu12 crystals?

The following table summarizes the data collection parameters used for Os4BGlu12 crystals:

|Parameter|Os4BGlu12|Os4BGlu12–DNP2FG|
|--|--|
|Beamline|BL13B1|BL13B1|
|Detector|ADSC Quantum 315 CCD|ADSC Quantum 315 CCD|
|Crystal-to-detector distance (nm)|300|280|
|Wavelength (Å)|1.00|1.00|
|Exposure time (s)|20|15|
|Resolution range (Å)|30–2.50 (2.50–2.49)|30–2.45 (2.51–2.45)|
|No. of unique reflections|39533|43131|
|Completeness (%)|100.0 (99.9)|100.0 (99.9)|
|Average redundancy per shell|9.7 (10.0)|8.4 (8.6)|
|〈I/σ(I)〉|19.6 (6.4)|21.7 (4.4)|
|Rmerge (%)†|10.6 (41.7)|9.4 (49.9)|

How does substrate binding affect the conformation of glucose in the active site?

Structural analysis of Os4BGlu12 in complex with different ligands has revealed interesting conformational changes in the glucose moiety:

• In the covalent intermediate with 2-deoxy-2-fluoroglucose (G2F), the glucose ring adopts a ⁴C₁ chair conformation

• In the non-covalently bound DNP2FG complex, the glucose ring assumes a ¹S₃ skew boat conformation

These observations are consistent with a hydrolysis mechanism proceeding via a ⁴H₃ half-chair transition state, providing valuable insights into the catalytic mechanism of Os4BGlu12 .

What is the proposed catalytic mechanism of Os4BGlu12?

Os4BGlu12 follows the classic retaining mechanism of glycoside hydrolases:

• The catalytic nucleophile (Glu393) attacks the anomeric carbon of the substrate, forming a covalent glycosyl-enzyme intermediate

• The glucose ring undergoes conformational changes during catalysis, transitioning through a ⁴H₃ half-chair conformation at the transition state

• The positioning of the catalytic nucleophile influences substrate specificity and reaction rate

The enzyme's ability to hydrolyze both O-glucosides and S-glucosides (though at a lower rate) suggests some flexibility in the active site that can accommodate different types of glycosidic bonds .

How does Os4BGlu12 differ functionally from other rice β-glucosidases?

Os4BGlu12 shows both similarities and differences when compared to other rice β-glucosidases:

• Unlike Os3BGlu6 and Os3BGlu7, Os4BGlu12 exhibits significant thioglucosidase activity

• The position of its catalytic nucleophile in the G2F structure more closely resembles that of Sinapsis alba myrosinase than other rice O-glucosidases

• It has distinct substrate preferences, particularly for oligosaccharide hydrolysis

These functional differences may reflect the diverse roles of β-glucosidases in plant physiology and stress responses .

What role might Os4BGlu12 play in plant stress responses?

While the search results don't directly address Os4BGlu12's role in stress responses, studies on homologous β-glucosidases provide insights. In Crocus sativus, CsBGlu12 transcript levels are highly induced by various stressors:

• UV-B exposure

• Dehydration

• NaCl (salt stress)

• Methyl jasmonate

• Abscisic acid

These findings suggest that BGlu12 enzymes may play important roles in plant stress responses, potentially through the release of bioactive compounds from their inactive glycosylated forms .

How might Os4BGlu12 contribute to antioxidant mechanisms in plants?

Studies on CsBGlu12 from Crocus sativus indicate that this homologous enzyme catalyzes the hydrolysis of flavonol β-glucosides, releasing flavonol aglycones with antioxidant properties. Transient overexpression of CsBGlu12 in Nicotiana benthamiana led to:

• Accumulation of antioxidant flavonols

• Enhanced tolerance to abiotic stresses

• Reduced levels of reactive oxygen species (ROS) during stress conditions

Given the similarities between CsBGlu12 and Os4BGlu12, the rice enzyme might also be involved in releasing antioxidant compounds during stress responses, though specific studies on Os4BGlu12 in this context would be needed to confirm this hypothesis .

How can site-directed mutagenesis be applied to study Os4BGlu12 function?

Site-directed mutagenesis is a powerful approach for investigating enzyme function. For BGlu12 enzymes:

• Mutation of conserved catalytic glutamic acid residues (such as Glu200 and Glu414 in CsBGlu12) completely abolishes β-glucosidase activity, confirming their essential role in catalysis

• Similar mutations in Os4BGlu12 would help determine the precise contributions of specific residues to substrate specificity and catalytic efficiency

• Structure-guided mutagenesis could be used to engineer Os4BGlu12 variants with altered substrate preferences or improved catalytic properties

These approaches would provide mechanistic insights and potentially generate enzyme variants with useful biotechnological applications.

What crystallographic techniques are valuable for studying enzyme-substrate interactions in Os4BGlu12?

Several crystallographic approaches have proven valuable for studying Os4BGlu12:

• Co-crystallization with inhibitors like DNP2FG to capture non-covalent complexes

• Using mechanism-based inhibitors that form stable covalent intermediates, such as G2F

• Comparison of apo and ligand-bound structures to identify conformational changes upon substrate binding

• Analysis of active site geometry and interactions in different complex structures

These approaches have revealed important details about glucose ring conformations during catalysis and the positioning of the catalytic nucleophile in different functional states .