Crystal Structure of Family 14 Polysaccharide Lyase with pH-dependent Modes of Action

Kohei Ogura,Masayuki Yamasaki,Takashi Yamada,Bunzo Mikami,Wataru Hashimoto,Kousaku Murata

doi:10.1074/jbc.m109.068056

Kohei Ogura, Masayuki Yamasaki + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.068056

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Abstract

The Chlorella virus enzyme vAL-1 (38 kDa), a member of polysaccharide lyase family 14, degrades the Chlorella cell wall by cleaving the glycoside bond of the glucuronate residue (GlcA) through a beta-elimination reaction. The enzyme consists of an N-terminal cell wall-attaching domain (11 kDa) and a C-terminal catalytic module (27 kDa). Here, we show the enzyme characteristics of vAL-1, especially its pH-dependent modes of action, and determine the structure of the catalytic module. vAL-1 also exhibited alginate lyase activity at alkaline pH, and truncation of the N-terminal domain increased the lyase activity by 50-fold at pH 7.0. The truncated form vAL-1(S) released di- to hexasaccharides from alginate at pH 7.0, whereas disaccharides were preferentially generated at pH 10.0. This indicates that vAL-1(S) shows two pH-dependent modes of action: endo- and exotypes. The x-ray crystal structure of vAL-1(S) at 1.2 A resolution showed two antiparallel beta-sheets with a deep cleft showing a beta-jelly roll fold. The structure of GlcA-bound vAL-1(S) at pH 7.0 and 10.0 was determined: GlcA was found to be bound outside and inside the cleft at pH 7.0 and 10.0, respectively. This suggests that the electric charges at the active site greatly influence the binding mode of substrates and regulate endo/exo activity. Site-directed mutagenesis demonstrated that vAL-1(S) has a specific amino acid arrangement distinct from other alginate lyases crucial for catalysis. This is, to our knowledge, the first study in which the structure of a family 14 polysaccharide lyase with two different modes of action has been determined.

Highlights

Insertion of viral DNA into the host cell, and (iv) release of amplified viruses out of the host cell [1,2,3]
The surface protein Vp130 encoded by the Chlorella virus CVK2 attaches to cell wall of the Chlorella strain NC64A [4]
The protein vAL-1 produced by the Chlorella virus CVK2 was identified to be a cell wall material (CWM)-degrading2 enzyme [10]. vAL-1 binds to Chlorella NC64A, recognizes ␣- or ␤-glucuronate (GlcA) in the CWM, and cleaves its glycoside bonds through ␤-elimination; in other words, vAL-1 acts as a CWM lyase [11] (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Materials—Synthetic oligonucleotides utilized for PCR were purchased from Hokkaido System Science. E. coli cells harboring pET42b-vAL-1 were grown in Luria-Bertani medium [21], collected, suspended in 0.5 M NaCl and 20 mM Tris-HCl (pH 7.5) (buffer A) including 0.5% Triton X-100, and ultrasonically disrupted. E. coli cells harboring the plasmid pET21b-vAL-1(S) were grown in LB medium, collected, suspended in buffer A including 0.5% Triton X-100, and ultrasonically disrupted. These cells produced a protein consisting of vAL-1(S) and a histidinetagged sequence at the C terminus. The specific activity of vAL-1, vAL-1(S), and the mutants was assayed with a reaction mixture containing 1.0 mg/ml sodium alginate, 200 mM Hepes (pH 7.0) or Gly (pH 10.0), and 0.1 mg/ml enzyme.

Data collection and refinement statistics

RESULTS AND DISCUSSION

Alginate lyase Glucuronan lyase A