Abstract
The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The 1.5 Å crystal structure reveals that Cellvibrio mixtus designated -mannosidase 5A (CmMan5A) adopts a (/␣)8 barrel fold, and superimposition with glycoside hydrolase family 5 (GH5) endomannanases shows that dramatic differences in the length of three loops modify the active center accessibility and modulate the specificity from endo to exo
Mannose-containing polysaccharides are an important component of the plant cell wall and are present mainly as galactomannans in which the 1,4-linked mannopyranoside backbone is decorated with galactosyl residues at the O-6 position and glucomannan that contains a heterogeneous backbone of 1,4-linked glucose and mannose sugars [1] (Fig. 1)
Summary
Bacterial Strains, Plasmids, and Culture Conditions—C. mixtus (NCIMB 8633) was cultured aerobically at 20 °C in Dubos mineral salts medium or on Dubos agar plates overlaid with filter paper [12]. The recombinant -mannosidase was expressed in E. coli BL21 (DE3; Novagen) containing a derivative of pET21a encoding CmMan5A and was cultured as described below. Plasmids (pBluescript SKϪ) containing genomic DNA inserts from C. mixtus were excised from positive recombinant phage and rescued into E. coli XL1Blue, as described in the Stratagene protocol. Expression and Purification of CmMan5A—To express wild type and mutant forms of CmMan5A in E. coli, DNA encoding the mature enzyme (residues 26 – 456) was amplified by PCR from C mixtus genomic DNA using the primers 5Ј-CTCCATATGGTTGCAGAAAGTAAC-3Ј and 5Ј-CACCTCGAGTTTCGGCTGAAAACG-3Ј (incorporated restriction sites are in bold type) and the thermostable DNA polymerase pFU Turbo. The recombinant of pGEM T-easy containing man5A was digested with NdeI and XhoI, and the excised mannosidase gene was cloned into the restricted expression vector pET21a to generate pFD1. The enzyme was buffer exchanged, using a PD-10 Sephadex G-25 M gel filtration column (Amersham Biosciences), into 50 mM sodium Hepes buffer, pH 7.5, containing 200 mM NaCl (Buffer A), concentrated to 20 mg mlϪ1 with Amicon 10-kDa molecular mass centrifugation membranes, and subjected to gel filtration using a HiLoad 16/60 Superdex 75 column (Amersham Biosciences) with protein eluted at 1 ml minϪ1 in Buffer A
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