Structure of the β‐glucosidase gene bglA of Clostridium thermocellum

Abstract

The nucleotide sequence of the Clostridium thermocellum gene bglA, coding for the thermostable j-glucosidase A, has been determined. The coding region of 1344 bp was identified by comparison with the N-terminal amino acid squence of recombinant b-glucosidase A purified from Escherichiu coli. The deduced amino acid sequence corresponds to a protein of 51482 Da. The coding region is flanked by putative promoter and transcription terminator sequences. The protein is unrelated to P-glucosidase B of C . thermocellz4m, but has a high level of similarity with other bacterial P-glucosidases and phospho-P-glucosidases. Similarity is also observed with the Bgalactosidase of the archaebacterium Sulfolobus solfuturicus. Unexpectedly, it was found that human lactasephlorizin hydrolase contains three copies of a sequence closely related to C. thermocellum P-glucosidase A (up to 40% sequence identity). These diverse 8-glucosidases can therefore be grouped into an enzyme family (BGA) of common structural design. Sequence comparison by hydrophobic cluster analysis revealed that all BGA enzymes share a well conserved region which is homologous to the catalytic domain of the widely distributed cellulase family A. A distinctive feature of this domain is the sequence motif His-Asn-Glu-Pro in which the catalytic residues His and Glu are separated by 35 -55 amino acid residues. The cellulase family A and the fl-glucosidase family BGA might thus be considered as members of a protein super-family comprising j-glucanases and flglycosidases from all three primary kingdoms of living organisms.P-Glucosidases (P-D-glucoside glucohydrolases) catalyze the hydrolysis of P-glycosidic bonds between glucose and aryl, alkyl, or saccharide groups. Most P-glucosidases exhibit a low specificity in regard to the aglycon portion of the substrate [l]. Many enzymes are also nonspecific with respect to the C4 configuration of the sugar moiety and hydrolyze both P-glucosides and B-galactosides [2]. A particular case of low substrate specificity is the mammalian enzyme lactase/phlorizin hydrolase (LPH) which contains two distinct Pglycosidases (b-galactosidase and aryl-P-glucosidase) within one polypeptide chain [3, 41. On the other hand, specificity is usually very high with respect to phosphorylation at the C6 Enzymes. Restriction endonucleases BumHT, EcoRI, EcoRV, HindIII, KpnI, PstI, PvuII (EC 3.1.21.4); S1 nuclease (EC 3.1.30.1); E. coli exonuclease III (EC 3.1.11.2); T4 DNA ligase (EC 6.5.1.1); E. coli DNA polymerase (EC 2.7.7.7); P-D-ghcosidase (cellobiase) (EC 3.2.1.21); P-D-galactosidase (lactase) (EC 3.2.1.23); phlorizin hydrolase (EC 3.2.1.62); 6-phospho-P-~-galactosidase (EC 3.2.1.85); 6-phospho-P-D-glucosidase (EC 3.2.1.86); endoglucanase (cellulase) (EC 3.2.1.4); lysozyme (EC 3.2.1.17).Note. The novel nucleotide sequence data publishcd here have been deposited with the EMBL sequence data bank and are available under the accession number X60268. position of the sugar residue. Thus, the bacterial phospho-Pglucosidases associated with the phosphoe...

Functional diversity and interactions between the repeat domains of rat intestinal lactase

“…LPH is synthesized as a single-chain 215-245 kDa precursor comprising four tandemly repeated domains [2][3][4][5][6][7][8]. These show sequence similarity with the type 1 family of glycosyl hydrolases (T1GH family), a family of proteins that includes single-domain enzymes present in micro-organisms (see [9,10] and references therein] and plants [11][12][13], and in the liver of mammals [14]. The T1GH family also comprises multi-domain proteins expressed in the intestine of non-mammalian vertebrates and in invertebrates, but their sequences have not been fully determined [15].…”

Molecular cloning and bacterial expression of a cDNA encoding furostanol glycoside 26‐O‐β‐glucosidase of Costus speciosus

“…CSF26G1 cDNA should encode one of the C. speciosus F26G isozymes which might exist as a minor protein in the rhizomes. I~-Glucosidases are known to fall into two distinct families (BGA and BGB) based on their structure similarities rather than their biological roles and substrate specificities [20,21]. The BGA family includes bacterial ~-glucosidases, archaebacterial I~-galactosidase, plant thioglucosidases, and human lactase-phlorizin hydrolase, while the BGB family includes fungal and rumen bacterial I~-glucosidases [18,21].…”

A sequence analysis of the β‐glucosidase sub‐family B

“…It is worth noting that the N-terminal region of the multiple alignment groups together the well known sequence motifs [2]. Legler et al [21] observed that the conduritol B-epoxi inhibitor bound to the Asp residue of the SDW motif in Aspergillus wentii.…”

“…His, Glu and Asp are the amino acids thought to be involved in enzymatic hydrolysis using an acid catalytic mechanism [2]. Because of their biotechnological importance, a number of j?-glucosidase genes have been cloned and attempts at organizing /3-glucosidase families and domains have been reported [3,4].…”