The microbiota of the mammalian intestine depend largely on dietary polysaccharides as energy sources. Most of these polymers are not degradable by the host, but herbivores can derive 70% of their energy intake from microbial breakdown--a classic example of mutualism. Moreover, dietary polysaccharides that reach the human large intestine have a major impact on gut microbial ecology and health. Insight into the molecular mechanisms by which different gut bacteria use polysaccharides is, therefore, of fundamental importance. Genomic analyses of the gut microbiota could revolutionize our understanding of these mechanisms and provide new biotechnological tools for the conversion of polysaccharides, including lignocellulosic biomass, into monosaccharides.

The crystal structures of a deglycosylated form of the egg-white glycoprotein avidin and of its complex with biotin have been determined to 2.6 and 3.0 A, respectively. The structures reveal the amino acid residues critical for stabilization of the tetrameric assembly and for the exceptionally tight binding of biotin. Each monomer is an eight-stranded antiparallel -barrel, remarkably similar to that of the genetically distinct bacterial analog streptavidin. As in streptavidin, binding of biotin involves a highly stabilized network of polar and hydrophobic interactions. There are, however, some differences. The presence of additional hydrophobic and hydrophilic groups in the binding site of avidin (which are missing in streptavidin) may account for its higher affinity constant. Two amino acid substitutions are proposed to be responsible for its susceptibility to denaturation relative to streptavidin. Unexpectedly, a residual N-acetylglucosamine moiety was detected in the deglycosylated avidin monomer by difference Fourier synthesis.The biotin-binding proteins avidin (from egg white) and streptavidin (from the bacterium Streptomyces avidinii) occupy a place of honor in many fields of biology. The reason for interest in these proteins is 2-fold: (i) both proteins exhibit the highest known affinity (Ka 1015 M-1) in nature between a ligand and a protein (1), and (ii) largely as a consequence, the avidin-biotin (and streptavidin-biotin) system has been widely applied as a universal tool, particularly for diagnostic purposes (2).Several groups have tried to crystallize egg-white avidin (3-5) with only limited success. During the course of our studies, which involved chemical and physical properties of avidin, we succeeded in isolating an active deglycosylated form of this protein (6), the structure of which we report here.* Interestingly, the x-ray structure of the related, naturally nonglycosylated, bacterial protein streptavidin has already been determined (7,8). Comparison of these two genetically remote structures permits us to decipher the crucial elements for formation of such a strong binding site. It had been noted (9) that the primary structures of the two proteins are similar (see Fig. 1) and that the conserved amino acid residues are mostly confined to six homologous segments (10, 11). The current study has revealed that the major structural elements are also conserved and critical functional groups are retained in the binding site. Nevertheless, there are some notable differences in their properties, many of which can be explained in terms of the three-dimensional structures of avidin and streptavidin. MATERIALS AND METHODSCrystallization and Data Collection. "Lite avidin" (i.e., avidin with most of the oligosaccharide chain removed) wasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S. University), which had been determined by molecular replacement using streptavidin as a search ...

The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.

Greater understanding of the mechanisms contributing to chemical and enzymatic solubilization of plant cell walls is critical for enabling cost-effective industrial conversion of cellulosic biomass to biofuels. Here, we report the use of correlative imaging in real time to assess the impact of pretreatment, as well as the resulting nanometer-scale changes in cell wall structure, upon subsequent digestion by two commercially relevant cellulase systems. We demonstrate that the small, noncomplexed fungal cellulases deconstruct cell walls using mechanisms that differ considerably from those of the larger, multienzyme complexes (cellulosomes). Furthermore, high-resolution measurement of the microfibrillar architecture of cell walls suggests that digestion is primarily facilitated by enabling enzyme access to the hydrophobic cellulose face. The data support the conclusion that ideal pretreatments should maximize lignin removal and minimize polysaccharide modification, thereby retaining the essentially native microfibrillar structure.

The crystal structure of a family‐III cellulose‐binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine‐stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface‐exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.