Heparinase I from Flavobacterium heparinum: The Role of the Cysteine Residue in Catalysis as Probed by Chemical Modification and Site-Directed Mutagenesis

Sasisekharan, Ram; Leckband, Deborah; Godavarti, Ranga; Venkataraman, Ganesh; Cooney, Charles L.; Langer, Robert

doi:10.1021/bi00044a022

Cited by 40 publications

(52 citation statements)

References 24 publications

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“…The primers 2 for the H203A mutation were 5Ј-AATATCGCCGCTGATAAAGTT-3Ј and 5Ј-AACTTTATCAGCGGC-GATATT-3Ј and for the H203D mutation were 5Ј-AATATCGCCGAT-GATAAAGTT-3Ј and 5Ј-AACTTTATCATCGGCGATATT-3Ј. The mutant genes were cloned into pET-15b and were sequenced essentially as described by Sasisekharan et al (1995).…”

Section: Mutagenesis Expression and Purification Of R-heparinase Imentioning

confidence: 99%

“…While there are several reports on the substrate specificity of heparinases (Rice and Linhardt, 1989;Linhardt et al, 1990;Nader et al, 1990;Desai et al, 1993;Ernst et al, 1995), there is limited information on the molecular features of the enzymes that confer this specificity (Linhardt et al, 1986;Lohse and Linhardt, 1992;Sasisekharan et al, 1995;Ernst et al, 1995).…”

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Heparinase I from Flavobacterium heparinum

Sasisekharan

Venkataraman

Godavarti

et al. 1996

Journal of Biological Chemistry

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In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (K d of 60 nM) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.

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Section: Mutagenesis Expression and Purification Of R-heparinase Imentioning

confidence: 99%

mentioning

confidence: 99%

Heparinase I from Flavobacterium heparinum

Sasisekharan

Venkataraman

Godavarti

et al. 1996

Journal of Biological Chemistry

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“…To facilitate purification, the heparinase I gene was expressed using the pET-15b system (Novagen). This construct has a polyhistidine tag and a thrombin cleavage site in a 21-amino acid N-terminal leader sequence (14).…”

Section: Mutagenesis Expression and Purification Of R-heparinase Imentioning

confidence: 99%

“…All the mutant genes were cloned into pET-15b and were sequenced to verify the mutations, as described previously (14).…”

Section: Mutagenesis Expression and Purification Of R-heparinase Imentioning

confidence: 99%

“…We subsequently carried out extensive biochemical studies to investigate structure-activity relationships of heparinase I and to understand the mechanism of heparin degradation (14 -16). Earlier we showed that cysteine 135, in a highly positively charged environment, is catalytically active in heparinase I (14). We proposed that one possible role of the positively charged active site would be to lower the pK a of the cysteine such that it is present as a thiolate anion in the enzyme active site.…”

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Heparinase I from Flavobacterium heparinum

Godavarti¹,

Sasisekharan

1998

Journal of Biological Chemistry

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Heparinases are bacterial enzymes that are powerful tools to study the physiological roles of heparin-like complex polysaccharides. In addition, heparinases have significant therapeutic applications. We had proposed earlier that cysteine 135 and histidine 203 together form the catalytic domain in heparinase I. We had also identified a heparin binding domain in heparinase I containing two positively charged clusters HB-1 and HB-2 in a primary heparin binding site and other positively charged residues in the vicinity of cysteine 135. In this study, through systematic site-directed mutagenesis studies, we show that the alteration of the positive charge of the HB-1 region has a pronounced effect on heparinase I activity. More specifically, site-directed mutagenesis of K199A (contained in HB-1) results in a 15-fold reduction in catalytic activity, whereas a K198A mutation (also in HB-1) results in only a 2-to 3-fold reduction in heparinase I activity. A K132A mutation, in close proximity to cysteine 135, also resulted in reduced (8-fold) activity. Heparin affinity chromatography experiments indicated moderately lowered binding affinities for the K132A, K198A, and the K199A mutant enzymes. The above results, taken together with our previous observations, lead us to propose that the positively charged heparin binding domain provides the necessary microenvironment for the catalytic domain of heparinase I. The dominant effect of lysine 199 suggests an additional, more direct, role in catalysis for this residue. Heparin-like glycosaminoglycans (HLGAGs)1 play an intricate role in the extracellular matrix, regulating a wide variety of biological functions (1, 2). HLGAGs are highly sulfated, complex, acidic polysaccharides consisting of alternating uronic acid (L-iduronic or D-glucuronic acid) and D-glucosamine residues connected through 1-4 linkages. Variations in the degree and distribution of sulfation result in a high degree of chemical heterogeneity in HLGAGs. Three enzymes that degrade HLGAGs (heparin and heparan sulfate), viz. heparinases I, II, and III from Flavobacterium heparinum, recognize unique sequences of sulfation and uronic acid epimerization in HLGAGs with a high degree of specificity (3-5). Heparinases have important clinical applications such as in the monitoring of heparin levels in blood (approved by the FDA) (6), neutralization of heparin in blood (in phase III clinical trials), and in production of low molecular weight heparins for use in humans. In addition, heparinases I and III are potent inhibitors of neovascularization (7). More importantly, heparinases have proven to be useful tools in understanding the important physiological roles of HLGAGs (8, 9).Our group has cloned, sequenced, and expressed in Escherichia coli the genes for heparinases I, II, and III from F. heparinum (10 -13). We subsequently carried out extensive biochemical studies to investigate structure-activity relationships of heparinase I and to understand the mechanism of heparin degradation (14 -16). Earlier we showed that cystei...

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Biochemical analysis and kinetic modeling of the thermal inactivation of MBP‐fused heparinase I: Implications for a comprehensive thermostabilization strategy

Chen

et al. 2011

Biotech & Bioengineering

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Enzymatic degradation of heparin by heparin lyases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin (LMWH). However, the commercial use of heparinase I (HepI), one of the most studied heparin lyases, has been largely hampered by its low productivity and extremely poor thermostability. Here we report the thermal inactivation mechanism and strategic thermal stabilization of maltose-binding protein (MBP)-HepI, a fusion HepI produced in E. coli with high yield, solubility and activity. Biochemical studies demonstrated that the thermal inactivation of MBP-HepI involves an unfolding step that is temperature-dependently reversible, followed by an irreversible dimerization step induced by intermolecular disulfide bonds. A good consistency between the kinetic modeling and experimental data of the inactivation was obtained within a wide range of temperature and enzyme concentration, confirming the adequacy of the proposed inactivation model. Based on the inactivation mechanism, a comprehensive strategy was proposed for the thermal stabilization of MBP-HepI, in which Ca(2+) and Tween 80 were used to inhibit unfolding while site mutation at Cys297 and DTT were employed to suppress dimerization. The engineered enzyme exhibits remarkably improved storage and operational thermostability, for example, 16-fold increase in half-life at its optimum temperature of 30 °C and 8-fold increase in remaining activity of 95% after 1-week storage at 4 °C, and therefore shows great potential as a commercial biocatalyst for heparin degradation in the pharmaceutical industry.

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Heparinase I from Flavobacterium heparinum: The Role of the Cysteine Residue in Catalysis as Probed by Chemical Modification and Site-Directed Mutagenesis

Cited by 40 publications

References 24 publications

Heparinase I from Flavobacterium heparinum

Heparinase I from Flavobacterium heparinum

Heparinase I from Flavobacterium heparinum

Biochemical analysis and kinetic modeling of the thermal inactivation of MBP‐fused heparinase I: Implications for a comprehensive thermostabilization strategy

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