Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum.

BACKGROUND: Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster. RESULTS: Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw. CONCLUSIONS: This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Important Structural Features of Thiolate-Rich Four-Helix Bundles for Cu(I) Uptake and Removal.

A family of bacterial copper storage proteins (the Csps) possess thiolate-lined four-helix bundles whose cores can be filled with Cu(I) ions. The majority of Csps are cytosolic (Csp3s), and in vitro studies carried out to date indicate that the Csp3s from Methylosinus trichosporium OB3b (MtCsp3), Bacillus subtilis (BsCsp3), and Streptomyces lividans (SlCsp3) are alike. Bioinformatics have highlighted homologues with potentially different Cu(I)-binding properties from these characterized "classical" Csp3s. Determination herein of the crystal structure of the protein (RkCsp3) from the methanotroph Methylocystis sp. strain Rockwell with Cu(I) bound identifies this as the first studied example of a new subgroup of Csp3s. The most significant structural difference from classical Csp3s is the presence of only two Cu(I) sites at the mouth of the bundle via which Cu(I) ions enter and leave. This is due to the absence of three Cys residues and a His-containing motif, which allow classical Csp3s to bind five to six Cu(I) ions in this region. Regardless, RkCsp3 exhibits rapid Cu(I) binding and the fastest measured Cu(I) removal rate for a Csp3 when using high-affinity ligands as surrogate partners. New experiments on classical Csp3s demonstrate that their His-containing motif is not essential for fast Cu(I) uptake and removal. Other structural features that could be important for these functionally relevant in vitro properties are discussed.

Turning a potent family-9 free cellulase into an operational cellulosomal component and vice versa.

Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family-9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, whereas its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In this study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C-terminal moiety of Cel9A encompassing the two X2 modules and the family-3b carbohydrate binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family-48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family-9 cellulase.