Substrate recognition by recombinant serine collagenase 1 from Uca pugilator

Area of Science:

• Biochemistry
• Enzymology
• Crustacean Biology

Background:

• Collagenases are crucial enzymes involved in extracellular matrix degradation.
• Serine proteases, including chymotrypsin, play diverse biological roles.
• Understanding crab collagenases provides insights into invertebrate protease function.

Purpose of the Study:

• To clone and characterize serine collagenase 1 from Uca pugilator.
• To investigate the enzymatic properties and substrate specificity of the recombinant enzyme.
• To compare the catalytic mechanism of crab collagenase with other serine proteases.

Main Methods:

• Cloning and sequencing of Uca pugilator serine collagenase 1 from a hepatopancreas cDNA library.
• Expression of the zymogen form in Saccharomyces cerevisiae as an alpha-factor fusion protein.
• Activation with trypsin and characterization of collagenolytic activity and substrate binding.

Main Results:

• The full-length sequence encodes a 270-amino acid pre-pro-enzyme similar to chymotrypsin family members.
• Recombinant collagenase showed identical collagenolytic properties to the native enzyme.
• Substrate binding pocket prefers basic, hydrophobic, and neutral polar residues; beta-branched and acidic amino acids are poor substrates.
• Acylation, not deacylation, is rate-limiting for collagenase catalysis.
• Collagenase compensates for lower primary residue binding efficiency with effective binding of extended peptidyl residues.

Conclusions:

• Uca pugilator serine collagenase 1 is a novel member of the chymotrypsin protease family.
• The enzyme possesses a unique substrate binding pocket and catalytic mechanism.
• This study expands the understanding of serine protease diversity and function in invertebrates.