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Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana.

Wang Y, Vilaplana F, Brumer H, Aspeborg H - Planta (2013)

Bottom Line: However, the galactose-rich and highly branched guar gum was not as efficiently degraded.The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively.Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

View Article: PubMed Central - PubMed

Affiliation: Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre, 106 91, Stockholm, Sweden.

ABSTRACT
Each plant genome contains a repertoire of β-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana β-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 °C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co(2+) and inhibited by Mn(2+). The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

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Sequence alignment of AtMan5-1 together with the plant mannanases GmMAN1 and LeMAN4a. In addition, fungal sequences have been included from Chrysonilia sitophila, Aspergillus niger and Trichoderma reesei. Catalytic glutamates are marked with a star and other highly conserved GH5 amino acids are highlighted with a black filled circle. The arginine located at the +2 subsite is marked with a cross
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Fig2: Sequence alignment of AtMan5-1 together with the plant mannanases GmMAN1 and LeMAN4a. In addition, fungal sequences have been included from Chrysonilia sitophila, Aspergillus niger and Trichoderma reesei. Catalytic glutamates are marked with a star and other highly conserved GH5 amino acids are highlighted with a black filled circle. The arginine located at the +2 subsite is marked with a cross

Mentions: The AtMan5-1 protein consists of 411 amino acids. There are two predicted glycosylation sites according to the NetNGlyc 1.0 server, one at the N-terminus and the second one at the C-terminus, whereas the ELM program suggests a site at the C-terminus, although a different sequon as compared with the NetNGlyc prediction. By sequence alignment analysis the catalytic glutamates can easily be identified as (E198) and (E319), as well as other well conserved residues within the GH5 family (Fig. 2) (Jenkins et al. 1995; Henrissat et al. 1996). Most amino acids suggested to be involved in substrate binding of LeMAN4A seem to be maintained in AtMan5-1 (Dilokpimol et al. 2011; Bourgault et al. 2005). For instance, an arginine (R200) at the +2 subsite important for transglycosylation capability is conserved in AtMan5-1 (Rosengren et al. 2012). However, differences can also be observed. The LeMAN4a tryptophan (W135) and the glutamine (Q282) located at the +1 subsite are substituted with a phenylalanine and a serine, respectively, whereas a serine (S369) and a phenylalanine (F370) positioned around subsite −3 in the LeMAN4a structure have been replaced with two leucines in AtMan5-1 (Fig. 2). Notably, all of the Brassicaceae GH5_7 proteins in the five-membered outgroup clade including AtMan5-1 are lacking an aromatic amino acid at the position corresponding to (F370) in the protein alignment.Fig. 2


Enzymatic characterization of a glycoside hydrolase family 5 subfamily 7 (GH5_7) mannanase from Arabidopsis thaliana.

Wang Y, Vilaplana F, Brumer H, Aspeborg H - Planta (2013)

Sequence alignment of AtMan5-1 together with the plant mannanases GmMAN1 and LeMAN4a. In addition, fungal sequences have been included from Chrysonilia sitophila, Aspergillus niger and Trichoderma reesei. Catalytic glutamates are marked with a star and other highly conserved GH5 amino acids are highlighted with a black filled circle. The arginine located at the +2 subsite is marked with a cross
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928506&req=5

Fig2: Sequence alignment of AtMan5-1 together with the plant mannanases GmMAN1 and LeMAN4a. In addition, fungal sequences have been included from Chrysonilia sitophila, Aspergillus niger and Trichoderma reesei. Catalytic glutamates are marked with a star and other highly conserved GH5 amino acids are highlighted with a black filled circle. The arginine located at the +2 subsite is marked with a cross
Mentions: The AtMan5-1 protein consists of 411 amino acids. There are two predicted glycosylation sites according to the NetNGlyc 1.0 server, one at the N-terminus and the second one at the C-terminus, whereas the ELM program suggests a site at the C-terminus, although a different sequon as compared with the NetNGlyc prediction. By sequence alignment analysis the catalytic glutamates can easily be identified as (E198) and (E319), as well as other well conserved residues within the GH5 family (Fig. 2) (Jenkins et al. 1995; Henrissat et al. 1996). Most amino acids suggested to be involved in substrate binding of LeMAN4A seem to be maintained in AtMan5-1 (Dilokpimol et al. 2011; Bourgault et al. 2005). For instance, an arginine (R200) at the +2 subsite important for transglycosylation capability is conserved in AtMan5-1 (Rosengren et al. 2012). However, differences can also be observed. The LeMAN4a tryptophan (W135) and the glutamine (Q282) located at the +1 subsite are substituted with a phenylalanine and a serine, respectively, whereas a serine (S369) and a phenylalanine (F370) positioned around subsite −3 in the LeMAN4a structure have been replaced with two leucines in AtMan5-1 (Fig. 2). Notably, all of the Brassicaceae GH5_7 proteins in the five-membered outgroup clade including AtMan5-1 are lacking an aromatic amino acid at the position corresponding to (F370) in the protein alignment.Fig. 2

Bottom Line: However, the galactose-rich and highly branched guar gum was not as efficiently degraded.The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively.Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

View Article: PubMed Central - PubMed

Affiliation: Division of Glycoscience, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Centre, 106 91, Stockholm, Sweden.

ABSTRACT
Each plant genome contains a repertoire of β-mannanase genes belonging to glycoside hydrolase family 5 subfamily 7 (GH5_7), putatively involved in the degradation and modification of various plant mannan polysaccharides, but very few have been characterized at the gene product level. The current study presents recombinant production and in vitro characterization of AtMan5-1 as a first step towards the exploration of the catalytic capacity of Arabidopsis thaliana β-mannanase. The target enzyme was expressed in both E. coli (AtMan5-1e) and P. pastoris (AtMan5-1p). The main difference between the two forms was a higher observed thermal stability for AtMan5-1p, presumably due to glycosylation of that particular variant. AtMan5-1 displayed optimal activity at pH 5 and 35 °C and hydrolyzed polymeric carob galactomannan, konjac glucomannan, and spruce galactoglucomannan as well as oligomeric mannopentaose and mannohexaose. However, the galactose-rich and highly branched guar gum was not as efficiently degraded. AtMan5-1 activity was enhanced by Co(2+) and inhibited by Mn(2+). The catalytic efficiency values for carob galactomannan were 426.8 and 368.1 min(-1) mg(-1) mL for AtMan5-1e and AtMan5-1p, respectively. Product analysis of AtMan5-1p suggested that at least five substrate-binding sites were required for manno-oligosaccharide hydrolysis, and that the enzyme also can act as a transglycosylase.

Show MeSH
Related in: MedlinePlus