Pedro M. Coutinho

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana

The synthesis, modification, and breakdown of carbohydrates is one of the most fundarnentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases ) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1–2% of an organism’s genes to glycoside hydrolases and glycosyltransferases alone. In plants, these processes are of particular importance for cell-wall synthesis and expansion, starch metabolism, defence against pathogens, symbiosis and signalling. Here we present an analysis of over 730 open reading frarnes representing the two main c1asses of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, in the genome of Arabidopsis thaliana. The vast importance of these enzymes in cell-wall formation and degradation is revealed along with the unexpected dominance of pectin degradation in Arabidopsis, with at least 170 open-reading frarnes dedicated solely to this task.

Glycogen metabolism loss: a common marker of parasitic behaviour in bacteria?

We searched 55 completely sequenced bacterial genomes for glycogen synthesis and degradation enzymes. A significant proportion of these bacteria appears to lack glycogen metabolism capability. Interestingly, these bacteria are parasitic, symbiotic or fastidious (i.e. difficult to culture outside their normal environment). It is suggested that the lack of bacterial glycogen metabolism is a trait associated with parasitic behaviour in bacteria.

Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in glycoside hydrolase family 1.

The Sphingomonas paucimobilis beta-glucosidase Bgl1 is encoded by the bgl1 gene, associated with an 1308 bp open reading frame. The deduced protein has a potential signal peptide of 24 amino acids in the N-terminal region, and experimental evidence is consistent with the processing and export of the Bgl1 protein through the inner membrane to the periplasmic space. A His(6)-tagged 44.3 kDa protein was over-produced in the cytosol of Escherichia coli from a recombinant plasmid, which contained the S. paucimobilis bgl1 gene lacking the region encoding the putative signal peptide. Mature beta-glucosidase Bgl1 is specific for aryl-beta-glucosides and has no apparent activity with oligosaccharides derived from cellulose hydrolysis and other saccharides. A structure-based alignment established structural relations between S. paucimobilis Bgl1 and other members of the glycoside hydrolase (GH) family 1 enzymes. At subsite -1, the conserved residues required for catalysis by GH1 enzymes are present in Bgl1 with only minor differences. Major differences are found at subsite +1, the aglycone binding site. This alignment seeded a sequence-based phylogenetic analysis of GH1 enzymes, revealing an absence of horizontal transfer between phyla. Bootstrap analysis supported the definition of subfamilies and revealed that Bgl1, the first characterized beta-glucosidase from the genus Sphingomonas, represents a very divergent bacterial subfamily, closer to archaeal subfamilies than to others of bacterial origin.

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase

Abstract The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCCxa031461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser153 (within the G-X-S-X-G consensus sequence), His75 and Asp125. The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641xa0Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pHxa07.4, optimal temperature 37xa0°C).