O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase are also found in human pathogens and symbionts. We report the three-dimensional structure of O-GlcNAcase from the human gut symbiont Bacteroides thetaiotaomicron both in its native form and in complex with a mimic of the reaction intermediate. Mutagenesis and kinetics studies show that the bacterial enzyme, very similarly to its human counterpart, operates via an unusual 'substrate-assisted' catalytic mechanism, which will inform the rational design of enzyme inhibitors.

Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.

Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module. CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families. Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available. We have determined the crystal structure of this module to a resolution of 2.1 Å. The protein is a ␤-sandwich that contains two potential ligand-binding clefts designated cleft A and B. The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site. The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6. These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Enzyme-catalyzed ␤-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a ''family PL-10'' polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 Å, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the ''parallel ␤-helix'' topology. The ''Michaelis'' complex of an inactive mutant in association with the substrate trigalacturonate͞Ca 2؉ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the ؊1 and ؉1 subsite saccharide carboxylate groups by a protein-liganded Ca 2؉ ion, the positioning of an arginine catalytic base in close proximity to the ␣-carbon hydrogen and numerous other conserved enzyme-substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-␤-elimination mechanism.