Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253

Abstract

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252^T (homolog strain). The analysis showed that in the homolog strain DSM 4252^T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.

Figure 1

Gene cluster analysis in Rhodothermus marinus DSM 4253. The gene cluster corresponding to GH3 loci in strains DSM 4252^T and DSM 4253 is identical. (A) Downstream of Rmar_0536 are genes enconding: membrane proteins (yedZ and yedY); a membrane transporter protein (mfs); an arylesterase precursor (arylest.); an ABC transporter ATP-binding protein (yvcR); a hypothetical protein (hyp.). Upstream of Rmar_0536 are genes encoding: a DNA-binding protein (hiran); two hypothetical proteins (hyp.); a potential methyltransferase (duf43). (B) Downstream of Rmar_0925 are genes encoding: a methylmalonyl-CoA mutase (mm_coa_mut); a 1,4-dihydroxy-2-naphthoyl-CoA synthase (menB); a 4′-phosphopantetheinyl transferase (sfp); a superoxide dismutase (sodA); a hypothetical protein (hyp.). Upstream of Rmar_0925 are genes encoding: ATPase (atpase); a 23 S rRNA methyltransferase (rlmN); an O-acetylhomoserine aminocarboxypropyltransferase (acpt); a homoserine O-acetyltransferase (esterase). (C) Downstream of Rmar_1080 and Rmar_1081 are genes encoding: two hypothetical proteins (hyp.); a periplasmic ABC transporter substrate-binding protein (abc), a potential cobalamin binding protein (yvcR.); a cobalamin biosynthesis protein (cbiX); a heme-binding protein (hmuY). Upstream of Rmar_1080 and Rmar_1081 genes encoding: an α-Glucuronidase (α-glucuronidase) and a pair of susC (a TonB-dependent receptor) and susD homologes. (D) Downstream of Rmar_2069 are genes encoding: a tRNA-Gln-CTG,tRNA (gray arrow); a hypothetical protein similar to ABC transporter (hyp. abc); a hypothetical protein (hyp.); a riboflavin synthase (ribD); a phosphohydrolase (hd); a riboflavin. Upstream of Rmar_2069 are genes encoding: an asparagine synthase (asn_synth); a Fe-S oxidoreductase (rad sam); a FAD-dependent oxidoreductase (oxidoreduct.); a hypothetical protein (hyp.). (E) Downstream of Rmar_2616 are genes encoding: a glycoside hydrolase of the GH43_62_32_68 superfamily (gh43_62_32_68); a predicted glycosyl hydrolase of the GH43/DUF377 family (gh43); a protein similar to glycosyltransferase involved in cell wall biosynthesis (rfaB); a short-chain dehydrogenase (sdr); a saccharopine dehydrogenas (sacc). Upstream of Rmar_2616 are genes encoding: a RNA polymerase ECF-type sigma factor gene (sig70); a tRNA (cytosine-5-)-methyltransferase (met_tra); a Fmu domain protein (fmu); a flavin containing amine oxidoreductase (amine oxidase); a bacterioferritin (ferr.); an exodeoxyribonuclease III (exoIII).

Figure 2

Phylogenetic relationship between proteins in GH3. The maximum likelihood phylogenetic tree was calculated using amino acid sequences of biochemically characterized proteins from GH3 together with six protein sequences from Rhodothermus marinus (highlighted in colored boxes). Sequences belonging to particular group are colored in the same color and the schematic representation of domain organization is shown in a circle above the tree. If domain organization of particular protein differs from prevailing domain organization of the group to which it belongs, its domain organization is shown separately next to the protein.

Figure 3

Homology models of the six GH3 enzymes from Rhodothermus marinus DSM 4253. Ribbon representation of (A) RmBgl3A, (B) RmBgl3B, (C) RmBgl3C, (D) RmXyl3A, (E) RmXyl3B and (F) RmNag3. Domain 1 is colored in gray, domain 2 in purple, FnIII in blue, PA14 in green and the linker between domain 2 and FnIII in yellow. RmBgl3A, RmBgl3B and RmBgl3C were modelled as dimers, one chain in each dimer is colored in light colors while the other is colored in dark colors. The β-lactamase domain of RmNag3 was not modelled and is not represented in the figure.

Figure 4

Comparison of active sites of the β-glucosidase and β-xylosidases showing residues involved in (A) subsite −1 and (B) subsite +1. Superimposition of homology models of RmBgl3A (pink), RmBgl3B (blue), RmBgl3C, RmXyl3A (yellow) and RmXyl3B (orange). Cellobiose (gark gray) and laminaribiose (light gray) in subsite −1 and +1 from PDB entries 1IEX and 1J8V respectively, both solved in complex with ExoI from barley. RmXyl3A and RmXyl3B have similar −1 and +1 subsites and RmBgl3C have a similar −1 subsite as RmBgl3A in terms of possible interacting residues.