Characterization of glycosyl hydrolase family 3 β-N-acetylglucosaminidases from Thermotoga maritima and Thermotoga neapolitana

Abstract

The genes encoding beta-N-acetylglucosaminidase (nagA and cbsA) from Thermotoga maritima and Thermotoga neapolitana were cloned and expressed in Escherichia coli in order to investigate whether Thermotoga sp. is capable of utilizing chitin as a carbon source. NagA and CbsA were purified to homogeneity by HiTrap Q HP and Sephacryl S-200 HR column chromatography. Both enzymes were homodimers containing a family 3 glycoside hydrolase (GH3) catalytic domain, with a monomer molecular mass of 54 kDa. The optimal temperatures and pHs for the activities of the beta-N-acetylglucosaminidases were found to be 65-75 degrees C and 7.0-8.0, respectively. Both enzymes hydrolyzed chitooligomers such as di-N-acetylchitobiose and tri-N-acetylchitotriose, and synthetic substrates such as p-nitrophenyl-beta-D-glucose (pNPGlc), p-nitrophenyl N-acetyl beta-D-glucosamine (pNPGlcNAc), p-nitrophenyl di-N-acetyl beta-D-chitobiose (pNPGlcNAc(2)) and p-nitrophenyl tri-N-acetyl beta-D-chitotriose (pNPGlcNAc(3)). However, the enzymes had no activity against p-nitrophenyl-beta-D-galactose (pNPGal) and p-nitrophenyl N-acetyl beta-D-galactosamine (pNPGalNAc) or highly polymerized chitin. The k(cat) and K(m) values were determined for pNPGlcNAc, pNPGlcNAc(2) and pNPGlcNAc(3). The k(cat)/K(m) value for pNPGlcNAc was the highest among three synthetic substrates. NagA and CbsA initially hydrolyzed p-nitrophenyl substrates to give GlcNAc, suggesting that the enzymes have exo-activity with chitin oligosaccharides from the non-reducing ends, like other beta-N-acetylglucosaminidases. However, NagA and CbsA can be distinguished from other GH3-type beta-N-acetylglucosaminidases in that they are highly active against di-N-acetylchitobiose. Thus, the present results suggest that the physiological role of both enzymes is to degrade the chitooligosaccharides transported through membrane following hydrolysis of chitin into beta-N-acetylglucosamine to be further metabolized in Thermotoga sp.