Abstract
Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 A resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 A, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr(156), Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.
Highlights
Are engaged in normal cellular metabolic processes that involve the formation and breakage of glycosidic bonds along with glycosyl transferase [3]
GHs can be classified as exo- or endo-type of glycoside hydrolases that catalyze the hydrolysis of the glycosidic bond from the end or at the middle, respectively, of a polysaccharide chain
Active sites that consist of the key residues have been classified into three topologies by Davies and Henrissat [1]: (i) a pocket or a crater that preferentially recognizes a saccharide molecule with a non-reducing end, presenting the exo-type hydrolysis, (ii) a cleft or groove that accommodates a large substrate for endo-type cleavage, and (iii) a tunnel that enables a polysaccharide chain to be threaded through for efficient endohydrolase processivity
Summary
Expression and Purification of LPHase—The artificial LPHase gene without the sequence of signal peptide (the first 35 residues) was reconstructed by PCR using a set of primers (sequences not shown). The active fractions eluted in the range of 100 –250 mM NaCl were collected and concentrated 20-fold before loading onto a anion exchange HiTrap Q column (1.6 ϫ 20 cm, Amersham Biosciences), which was equilibrated with phosphate buffer (20 mM, pH 6.8). The SeMet-LPHase crystal belonged to space group P212121 with unit cell dimensions a ϭ 46.27, b ϭ 60.36, c ϭ 150.13, and one molecule per asymmetric unit. The LPHase complex structure was determined by molecular replacement methods using the apo-LPHase structure as a search model by MOLREP and refined by REFMAC5 coupled to ARP/wARP. The three best solutions were obtained until a root mean square tolerance of 1.5 Å This complex model was subjected to energy minimization using the Tripos force field [42] with the SYBYL 8.0 program (The Tripos Associates, St. Louis, MO)
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