The variable structural flexibility of the Bacillus circulans β-galactosidase isoforms determines their unique...

Area of Science:

• Enzymology
• Structural Biology
• Biotechnology

Background:

• β-Galactosidase (BgaD) from Bacillus circulans is crucial for synthesizing β-galactooligosaccharides (GOS).
• Isoform A (BgaD-A) possesses distinct synthetic properties compared to other isoforms, particularly isoform D (BgaD-D).

Purpose of the Study:

• To elucidate the structural differences between BgaD-A and BgaD-D.
• To understand how these structural variations influence the enzymatic activity and product specificity of BgaD-A.
• To provide insights for optimizing GOS production.

Main Methods:

• Cryoelectron microscopy (cryo-EM) was used to determine the structures of BgaD-A.
• X-ray crystal structure of BgaD-D was used for comparison.
• Transgalactosylation reactions were performed to analyze enzymatic activity and product selectivity.

Main Results:

• Substantial structural divergences were observed between BgaD-A and BgaD-D.
• BgaD-A possesses a flexible Big-4 domain and a novel 'barrier domain 8' that restricts access of longer substrates to the active site.
• BgaD-A demonstrated higher selectivity for shorter GOS production compared to BgaD-D.

Conclusions:

• The unique structural features of BgaD-A, particularly the barrier domain, dictate its substrate specificity.
• Understanding these structural determinants enhances our knowledge of β-galactosidase catalysis.
• This research has direct implications for the targeted production of specific β-galactooligosaccharides.