The Calcium-binding Sites of Heparinase I fromFlavobacterium heparinum Are Essential for Enzymatic Activity

Liu, Dongfang; Shriver, Zachary; Godavarti, Ranga; Venkataraman, Ganesh; Sasisekharan, Ram

doi:10.1074/jbc.274.7.4089

Cited by 31 publications

(21 citation statements)

References 18 publications

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“…Furthermore, through a combination of chemical modification and peptide mapping studies, we identify two calciumcoordinating motifs, with either one or both of these sites being important for the critical calcium-heparinase I interaction. In the accompanying study (14), we use the information derived herein to design a series of site-directed mutants of the calcium-binding sites of heparinase I to identify particular amino acids involved in calcium binding and critical for proper enzymatic functioning.…”

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confidence: 99%

Biochemical Investigations and Mapping of the Calcium-binding Sites of Heparinase I from Flavobacterium heparinum

Shriver

Liu

Hu³

et al. 1999

Journal of Biological Chemistry

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The heparinases from Flavobacterium heparinum are lyases that specifically cleave heparin-like glycosaminoglycans. Previously, amino acids located in the active site of heparinase I have been identified and mapped. In an effort to further understand the mechanism by which heparinase I cleaves its polymer substrate, we sought to understand the role of calcium, as a necessary cofactor, in the enzymatic activity of heparinase I. Specifically, we undertook a series of biochemical and biophysical experiments to answer the question of whether heparinase I binds to calcium and, if so, which regions of the protein are involved in calcium binding. Using the fluorescent calcium analog terbium, we found that heparinase I tightly bound divalent and trivalent cations. Furthermore, we established that this interaction was specific for ions that closely approximate the ionic radius of calcium. Through the use of the modification reagents N-ethyl-5-phenylisoxazolium-3-sulfonate (Woodward's reagent K) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, we showed that the interaction between heparinase I and calcium was essential for proper functioning of the enzyme. Preincubation with either calcium alone or calcium in the presence of heparin was able to protect the enzyme from inactivation by these modifying reagents. In addition, through mapping studies of Woodward's reagent Kmodified heparinase I, we identified two putative calcium-binding sites, CB-1 (Glu 207 -Ala 219 ) and CB-2 (Thr 373 -Arg 384 ), in heparinase I that not only are specifically modified by Woodward's reagent K, leading to loss of enzymatic activity, but also conform to the calciumcoordinating consensus motif.Heparin-like glycosaminoglycans, such as heparin and heparan sulfate, are acidic polysaccharides that play a role in many central biological processes, such as cell proliferation and signaling (1, 2). However, attempts to determine whether heparinlike glycosaminoglycans are involved in a particular biological process have been hampered by the lack of tools available to study these substrates.One such tool, under development in our laboratory, is the heparinases, bacterially derived lyases. Heparinase I from Flavobacterium heparinum is a 43-kDa enzyme that cleaves primarily heparin-like regions of heparin-like glycosaminoglycans (i.e. regions containing a high degree of sulfation with primarily iduronic acid as the uronic acid component) (3, 4). Heparinase I has been used in a variety of circumstances to highlight the importance of heparin-like glycosaminoglycans in such diverse biological processes as angiogenesis (5) and development (6).To extend the capabilities of the heparinases, we have undertaken a series of biochemical studies aimed at identifying important functional residues as well as elucidating the enzyme's mode of action. Through a combination of chemical modification, proteolytic mapping, and site-directed mutagenesis, we have identified Cys 135 (7), His 203 (8), and Lys 199 (9) as amino acid residues important for heparinase ...

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confidence: 99%

Biochemical Investigations and Mapping of the Calcium-binding Sites of Heparinase I from Flavobacterium heparinum

Shriver

Liu

Hu³

et al. 1999

Journal of Biological Chemistry

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“…The binding of Ca 2+ to alginate has been well documented (18,22), so it is possible that the true substrate for the epimerase reaction is the Ca 2+ -alginate complex. Finally, Ca 2+ is known to be a tightly bound cofactor in some enzymes (23,24). We therefore explored the Ca 2+ -dependence of the C5-mannuronan epimerase reaction.…”

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confidence: 99%

Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product

2005

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Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained 9 monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 s −1 to 0.0218 s −1 as the substrate size increased from 10 to 20 residues, and no additional increase in k cat was observed for substrates up to 100 residues in length. The K m decreased from 80 μM for substrate containing fewer than 15 residues to 4 μM for substrate containing over 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca 2+ for activity, and the Ca 2+ -alginate complex is not a substrate for the enzyme. Analysis of purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that 3 residues on the enzyme which all have a pK a of about 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1 H-NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.Pseudomonas aeruginosa is an opportunistic human pathogen that has been linked to certain pathological conditions of immunocompromised humans, and especially the respiratory tract infections that accompany cystic fibrosis (1) . Pulmonary infections caused by the mucoid phenotype of P. aeruginosa are almost impossible to eradicate, even with antibiotic treatment (2). The high antibiotic resistance is attributed in part to the formation of a biofilm, a complex extracellular polymeric matrix in which the cells are embedded. One of the primary constituents of the mature P. aeruginosa biofilm is the polysaccharide alginate. This cell-associated virulence factor is a high molecular weight (500-2000 kDa) linear polysaccharide comprised of residues of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), which are covalently linked by β-1,4 glycosidic bonds (3). The relative ratio of these building blocks in the polymer and the linear distribution of G residues strikingly alters physical properties of alginate such as viscosity and gel forming ability, and therefore plays a crucial role in the function of the biopolymer (4).Most of the genes required for alginate biosynthesis are located in the algD operon on the P. aeruginosa chromosome (5). The first polymeric product in the pathway is polymannuronan, which is synthesized from GDP-mannuronic acid. The formation of GDP-mannuronic acid from fructose...

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“…12) Recent experiments on site-directed mutagenesis have found that the calcium-binding sequences of heparinase I are essential for the enzyme activity. 27,28) In r-heparinase expression, the addition of 1 mM IPTG caused the formation of an inclusion body, an inactive form that is precipitated by centrifugation. Therefore, the induction was done by a low concentration of 0.05 mM IPTG, which induced a soluble active form of r-heparinase without formation of an inclusion body, but the expression level was lower than expected.…”

Section: Discussionmentioning

confidence: 99%

Cloning, Sequencing, and Expression of the Gene fromBacillus circulansThat Codes for a Heparinase That Degrades Both Heparin and Heparan Sulfate

Yoshida

Arakawa

Matsunaga

et al. 2002

Bioscience, Biotechnology, and Biochemistry

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The Calcium-binding Sites of Heparinase I fromFlavobacterium heparinum Are Essential for Enzymatic Activity

Cited by 31 publications

References 18 publications

Biochemical Investigations and Mapping of the Calcium-binding Sites of Heparinase I from Flavobacterium heparinum

Biochemical Investigations and Mapping of the Calcium-binding Sites of Heparinase I from Flavobacterium heparinum

Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product

Cloning, Sequencing, and Expression of the Gene fromBacillus circulansThat Codes for a Heparinase That Degrades Both Heparin and Heparan Sulfate

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