The plant enzyme hevamine has both chitinase and lysozyme activity. HPLC analysis of the products of the hydrolysis of chitopentaose shows that hevamine acts with retention of the configuration, despite the absence of a nucleophilic or stabilizing carboxylate. To analyze the stabilization of a putative oxocarbonium ion intermediate, the X-ray structure of hevamine complexed with the inhibitor allosamidin was determined at 1.85 A resolution. This structure supports the role of Glu127 as a proton donor. The allosamizoline group binds in the center of the active site, mimicking a reaction intermediate in which a positive charge at C1 is stabilized intramolecularly by the carbonyl oxygen of the N-acetyl group at C2.

Polygalacturonases specifically hydrolyze polygalacturonate, a major constituent of plant cell wall pectin. To understand the catalytic mechanism and substrate and product specificity of these enzymes, we have solved the x-ray structure of endopolygalacturonase II of Aspergillus niger and we have carried out site-directed mutagenesis studies. The enzyme folds into a right-handed parallel ␤-helix with 10 complete turns. The ␤-helix is composed of four parallel ␤-sheets, and has one very small ␣-helix near the N terminus, which shields the enzyme's hydrophobic core. Loop regions form a cleft on the exterior of the ␤-helix. The plant cell wall consists of a network of complex carbohydrates like cellulose, hemicellulose, and pectin. The latter is the most complex of these carbohydrates. It contains so-called "smooth regions" and "hairy regions." The smooth regions, also known as homogalacturonan, consist of ␣(1,4)-linked D-galacturonic acid residues, whereas the hairy regions, or rhamnogalacturonan I, are characterized by stretches of alternating ␣(1,2)-linked D-galacturonic acid and L-rhamnose (1). The rhamnose residues can be substituted at their O4 atoms by arabinose or galactose (2). Throughout the pectin molecule, the galacturonic acid residues can be methylated at O6 and/or acetylated at O2 and/or O3 (3). Due to its complex structure, modification of pectin by plants or complete breakdown by microorganisms requires many different enzymes.In microorganisms several classes of pectinases have been identified. These classes comprise pectate-, pectin-, and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases, and polygalacturonases, which all depolymerize the main chain; and pectin methylesterases and pectin-and rhamnogalacturonan acetylesterases, which act on the substituents of the main chain. Crystal structures are known of members of several classes of main chain depolymerizing pectinases. These include pectate lyases from Erwinia chrysanthemi and Bacillus subtilis (4 -6), pectin lyases from Aspergillus niger (7,8), and rhamnogalacturonase A from Aspergillus aculeatus (9). Recently, the crystal structure of endopolygalacturonase from the bacterium Erwinia carotovora was solved (10). The lyases cleave the substrate by ␤-elimination, whereas rhamnogalacturonases and polygalacturonases use acid/base-catalyzed hydrolysis (11,12). Despite their completely different reaction mechanisms, and their groupings in different sequence homology families, the x-ray structures of pectate lyase, pectin lyase, and rhamnogalacturonase reveal a similar unique right-handed parallel ␤-helix topology (13,14).

The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the ␤-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.Chitinases (EC 3.2.1.14) are glycosyl hydrolases which catalyze the degradation of chitin, an insoluble linear ␤-1,4-linked polymer of N-acetylglucosamine. Recently, chitinases have been receiving renewed attention because of their possible application for the biological control of chitin-containing organisms and also for the exploitation of natural chitinous materials. They are present in a wide range of organisms including bacteria, insects, viruses, plants, and animals and play important physiological and ecological roles. Chitinases so far sequenced are classified in two different families (namely, families 18 and 19) of the classification of glycosyl hydrolases based on amino acid sequence similarities (13,14). Family 18 contains chitinases from bacteria, fungi, viruses, and animals and class III and V chitinases from plants. On the other hand, class I, II, and IV plant chitinases belong to family 19, and this family solely comprises chitinases of plant origin. The two different families of chitinases display no sequence similarities with each other and have different three-dimensional structures (5).Oligosaccharides produced from partially acetylated chitosan by microbial chitinases have been previously studied in an attempt to clarify their specificity for ␤-1,4-N-acetylglucosaminic versus ␤-1,4-glucosaminic linkages (26, 27, 30). Except for chitinase C-1 from Streptomyces griseus HUT 6037, all of the examined microbial chitinases hydrolyzed GlcNAc-GlcNA...

The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P ‫؍‬ 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P ‫؍‬ 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P ‫؍‬ 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.Nucleic acid amplification assays (NAAAs) are commonly used in routine laboratories from industrialized countries for quick and specific detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Over the years, a significant improvement of PCR technologies has been achieved with the development of real-time PCR testing platforms. The main advantages of real-time PCR are a shortened turnaround time; automation of the procedure, which reduces hands-on time; and a decrease in the risk of cross-contamination (6). Recently, the GeneXpert system (Cepheid, Sunnyvale, CA), a real-time PCR that simultaneously detects both MTBC and rifampin resistance, was developed (1, 3, 9). In contrast to some real-time PCR instruments, the Xpert MTB/RIF is an on-demand assay described as a simple method that can be performed by personnel with minimal training and can provide results within 2 h (1, 3, 9). Recent studies (3, 9, 15, 16) reported a high sensitivity and specificity of the Xpert MTB/RIF test with respiratory specimens collected from patients living in countries with a high and a low prevalence of tuberculosis (TB). The detection of rifampin resistance, as a surrogate for multidrug-resistant TB (MDR-TB), directly from smear-positive respiratory specimens from patients having a high risk of MDR-TB has recently been recommended by the World Health...