Abstract
Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.
Highlights
Clostridium acetobutylicum, a bacterium used to produce acetone, butanol and ethanol from the fermentation of carbohydrates, is capable of using pectin as a feedstock (Schink et al, 1981)
Little is known about pectin degradation by C. acetobutylicum; the degradation of the polygalacturonan (PGA) and rhamnogalacturonan-I (RG-I) backbones of pectin by plant pathogens has been thoroughly studied, and many of the known pectinolytic enzymes share distant homology to putative gene products encoded by the C. acetobutylicum genome (Collmer & Keen, 1986; Prade et al, 1999; Ochiai et al, 2007; Marın-Rodrıguez et al, 2002; Nolling et al, 2001)
We identify CA_C0359 as a GH105 unsaturated glycoside hydrolase based on a structural analysis, with the potential to cleave the substrates of its homolog YteR and possibly other substrates
Summary
Clostridium acetobutylicum, a bacterium used to produce acetone, butanol and ethanol from the fermentation of carbohydrates, is capable of using pectin as a feedstock (Schink et al, 1981). Unsaturated glycoside hydrolases are intracellular proteins and can be divided into two carbohydrate-active enzyme (CAZy) families: GH105 and GH88. The core structure of GH105 hydrolases are closely related to those of GH88, they differ in substrate specificity and sequence identity (Collen et al, 2014; Itoh, Hashimoto et al, 2006b). GH105 proteins have been shown to cleave plant-based and algae-based substrates by hydrolyzing small two-chain and three-chain unsaturated polysaccharides containing an unsaturated uronyl saccharide at the nonreducing end (Yip & Withers, 2006; Garron & Cygler, 2010; Coutinho & Henrissat, 1999). Transcriptional studies and structural analysis of the CA_C0359-derived protein product presented here support its annotation as a GH105 family protein with an active pocket capable of binding unsaturated polysaccharides containing an unsaturated galacturonate. We identify CA_C0359 as a GH105 unsaturated glycoside hydrolase based on a structural analysis, with the potential to cleave the substrates of its homolog YteR and possibly other substrates
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