Mechanism of Hyaluronan Degradation byStreptococcus pneumoniae Hyaluronate Lyase

Abstract

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endoand exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra-and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52-and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn 349 , His 399 , and Tyr 408 , is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the ␣-and ␤-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.Streptococcus pneumoniae colonizes predominantly the upper respiratory tract of humans and is a major human pathogenic bacterium. It is one of the key causes of life-threatening disease such as pneumonia, bacteremia, and meningitis (1). It also causes less threatening diseases that are, however, very prevalent like otitis media and sinusitis (2). Pneumococci interact with the host and its tissues through the surface sugars (capsule) and a variety of usually surface-exposed protein molecules. These interactions are essential for the full pathogenicity of these bacteria and are likely involved in the diseasecausing processes. The proteins known to be involved in this interaction include, among others, hyaluronate lyase (3, 4), pneumolysin (5), pneumococcal surface protein A (6), and pneumococcal surface antigen A (7, 8).S. pneumoniae hyaluronate lyase (SpnHL) 1 primarily degrades hyaluronan (HA), the predominant polysaccharide component of animal and human connective tissues and the nervous system, into unsaturated disaccharide units as the end products (9). Cells in the connective tissues are embedded in the strikingly viscoelastic HA matrix. Hyaluronan is a polymeric glycan composed of linear repeats of a few hundred to as many as 20,000 or more disaccharide units of glucuronic acid and N-acetylglucosamine. The glycosidic linkage present within the disaccharide unit is ␤-1,3, whereas the disaccharide units are connected with the ␤-1,4-glycosidic linkage. The HA metabolism seems to be more si...

Superchannel of Bacteria: Biological Significance and New Horizons

“…Although the overall structure of the enzyme is basically the same as that of family PL-8 lyases for hyaluronate 39) and chondroitin ABC, 48) they differ in the loop structure over the cleft. Since PyrMan complexed with the enzyme is bound in the deep cleft, the active center (the substrate-binding site) is located in the cleft.…”

“…Other than these two regions, a region slightly conserved in family PL-7 lyases is found in -strand SA5, proximate to SA3 and SA4. Amino acid residues, including Tyr and His residues in SA3, SA4, and SA5, conserved in family PL-7 alginate lyases, are responsible for the catalytic actions of the enzymes, as in the cases of polysaccharide lyases such as family PL-5 alginate lyase A1-III, 37,38) and family PL-8 hyaluronate, 39) chondroitin, 40) and xanthan [45][46][47] lyases. In fact, His104, Tyr193, and Tyr199 of PA1167 constitute an active cleft.…”

“…In other polysaccharide lyases, such as hyaluronate and chondroitin lyases, two separate catalytic amino acid residues function in acid and base catalysis. 39,40) Catalysis by A1-III with a single Tyr as an active center can probably be regarded as a novelelimination reaction.…”

“…Although polysaccharide lyases are currently classified into 18 families (PL-1 to -18) on the basis of their primary structures, X-ray crystallography of them has indicated that almost all of them can be grouped into six folds: parallel -helix, lyases for pectate (families PL-1, -3, and -9), 49) and chondroitin B (family PL-6); 50) -sheet abundant flattened oval disk, rhamnogalacturonan lyase (family PL-4); 51) = -barrel, lyases for alginate (A1-III) (family PL-5) 36) and pectate (family PL-10); 52) = -barrel + anti-parallel -sheet, lyases for hyaluronate, 39) chondroitin AC, 40) and xanthan 46) (family PL-8);…”

“…-sandwich, alginate lyases PA1167 and A1-II 0 (family PL-7); 39) and -propeller, rhamnogalacturonan lyase YesW (family PL-11). 53) As for the = -barrel, we have confirmed that a porcine kidney bifunctional protein, renin-binding protein [N-acyl-D-glucosamine 2-epimerase (AGE)], 54,55) a novel glycosaminoglycan-degrading enzyme of unsaturated glucuronyl hydrolase (UGL) of Bacillus sp.…”