Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact n-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein

Role of ionic interactions and linker in the domain interaction and modulation of functional activity of hyaluronate lyases

Hyaluronate lyases from Streptococcus pneumoniae (SpnHL) and Streptococcus agalactiae (SagHL) are composed of four domains; N-terminal domain, spacer domain, α-domain and c-terminal domain, which are connected through peptide linkers. We have earlier shown that the recombinant α- and c-terminal domains of SpnHL/SagHL interact with each other even in absence of the linker and form a functional complex with enhanced enzymatic activity. Here, we looked into the role of ionic interactions in the enzyme stability and also the role of c-terminal domain and linker in the functional regulation. Domain swapping studies showed that the c-terminal domain does not bind directly to the substrate; instead the domain contributes to the interaction with the polymeric hyaluronan for catalysis. Furthermore, the substrate specificity exchanges with the size of catalytic cleft. The role of linker connecting α-domain to c-terminal domain was found to hold the c-terminal domain in a conformation suitable for achieving maximum activity

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (≥2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule

Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase

We have carried out a systematic study on the guanidinium chloride- and urea-induced unfolding of glucose oxidase from aspergillus niger, an acidic dimeric enzyme, using various optical spectroscopic techniques, enzymatic activity measurements, glutaraldehyde cross-linking, and differential scanning calorimetry. The urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the native dimeric enzyme molecule occurring in a single step. On the contrary, the GdmCl-induced unfolding of GOD was a multiphasic process with stabilization of a conformation more compact than the native enzyme at low GdmCl concentrations and dissociation along with unfolding of enzyme at higher concentrations of GdmCl. The GdmCl-stabilized compact dimeric intermediate of GOD showed an enhanced stability against thermal and urea denaturation as compared to the native GOD dimer. Comparative studies on GOD using GdmCl and NaCl demonstrated that binding of the Gdm+ cation to the enzyme results in stabilization of the compact dimeric intermediate of the enzyme at low GdmCl concentrations. An interesting observation was that a slight difference in the concentration of urea and GdmCl associated with the unfolding of GOD was observed, which is in violation of the 2-fold rule for urea and GdmCl denaturation of proteins. This is the first report where violation of the 2-fold rule has been observed for a multimeric protein

New anthraquinones from <i style="">Rubia cordifolia</i> roots

1945-1950Three new anthraquinones namely 1-hydroxy-2, 7-dimethylanthraquinone, 2-hydroxy-6-methylanthraquinone and 2, 6-dihydroxyanthraquinone along with n-nonadecane, n-heptadecane, 8'-hydroxy-n-pentadecanyl decan-4-en-1-oate, n-octacosanyl octa-1-oate and 3, 5-di-(p-hydroxybenzyl) phenol have been isolated from Rubia cordifolia roots and characterized on the basis of spectral data

Two new flavonoids from Adenium obesum grown in Oman

In this study, two new antimicrobial flavonoid compounds were isolated and characterized, and their biological activities were assayed. The pure antimicrobial compounds were isolated and characterized from the ethyl acetate extract using different chromatographic techniques. The antimicrobial activity of the isolated pure compounds was assessed using disk diffusion method. Phytochemical investigation on the ethyl acetate crude extract of stem of Adenium obesum (A. obesum) resulted in the isolation of two pure new flavonoids 5,7,3′,4′-tetrahydroxy flavone 1 and 3,5,7,3′,4′,5′-hexahydroxyflavone 2 with several other minor compounds. Their structures were deduced on the basis of 1H NMR, 13C NMR, DEPT 90 and 135, COXY, HMBC, and MS. The pure antimicrobial flavonoid compounds showed significant antibacterial activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris in the disk diffusion assay. Inhibition zones were 9–14 mm. The maximum inhibition was shown by compound 2 at concentration 200 μg/ml against P. vulgaris (IZ = 14 mm) when compared with the standard amoxicillin. The results showed that the isolated pure antimicrobial flavonoid compounds have significant antimicrobial activity, which can be used as antibiotics. This is the first report on isolation of these secondary metabolites from A. obesum