The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum -: A structural and biochemical study of the enzyme active site and saccharide substrate specificity

被引:20
作者
Raman, R
Myette, JR
Shriver, Z
Pojasek, K
Venkataraman, G
Sasisekharan, R
机构
[1] MIT, Div Biol Engn, Cambridge, MA 02139 USA
[2] MIT, Div Hlth Sci & Technol, Cambridge, MA 02139 USA
关键词
D O I
10.1074/jbc.M211425200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In the previous paper (Myette, J. R., Shriver, Z., Claycamp, C., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2003) J. Biol. Chem. 278,12157-12166), we described the molecular cloning, recombinant expression, and preliminary biochemical characterization of the heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. In this paper, we extend our structure-function investigation of the 2-O-sulfatase. First, we have constructed a homology-based structural model of the enzyme active site, using as a framework the available crystallographic data for three highly related arylsulfatases. In this model, we have identified important structural parameters within the enzyme active site relevant to enzyme function, especially as they relate to its substrate specificity. By docking various disaccharide substrates, we identified potential structural determinants present within these substrates that would complement this unique active site architecture. These determinants included the position and number of sulfates present on the glucosamine, oligosaccharide chain length, the presence of a Delta4,5-unsaturated double bond, and the exolytic versus endolytic potential of the enzyme. The predictions made from our model provided a structural basis of substrate specificity originally interpreted from the biochemical and kinetic data. Our modeling approach was further complemented experimentally using peptide mapping in tandem with mass spectrometry and site-directed mutagenesis to physically demonstrate the presence of a covalently modified cysteine (formylglycine) within the active site. This combinatorial approach of structure modeling and biochemical studies provides insight into the molecular basis of enzyme function.
引用
收藏
页码:12167 / 12174
页数:8
相关论文
共 23 条
[1]   1.3 Å structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family [J].
Boltes, I ;
Czapinska, H ;
Kahnert, A ;
von Bülow, R ;
Dierks, T ;
Schmidt, B ;
von Figura, K ;
Kertesz, MA ;
Usón, I .
STRUCTURE, 2001, 9 (06) :483-491
[2]   Structure of a human lysosomal sulfatase [J].
Bond, CS ;
Clements, PR ;
Ashby, SJ ;
Collyer, CA ;
Harrop, SJ ;
Hopwood, JJ ;
Guss, JM .
STRUCTURE, 1997, 5 (02) :277-289
[3]   Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine [J].
Dierks, T ;
Miech, C ;
Hummerjohann, J ;
Schmidt, B ;
Kertesz, MA ;
von Figura, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (40) :25560-25564
[4]   Molecular diversity of heparan sulfate [J].
Esko, JD ;
Lindahl, U .
JOURNAL OF CLINICAL INVESTIGATION, 2001, 108 (02) :169-173
[5]  
Higgins DG, 1996, METHOD ENZYMOL, V266, P383
[6]   A MOLECULAR MECHANICAL FORCE-FIELD FOR THE CONFORMATIONAL-ANALYSIS OF OLIGOSACCHARIDES - COMPARISON OF THEORETICAL AND CRYSTAL-STRUCTURES OF MAN-ALPHA-1-3MAN-BETA-1-4GLCNAC [J].
HOMANS, SW .
BIOCHEMISTRY, 1990, 29 (39) :9110-9118
[7]   FORCE-FIELD PARAMETERS FOR SULFATES AND SULFAMATES BASED ON AB-INITIO CALCULATIONS - EXTENSIONS OF AMBER AND CHARMM FIELDS [J].
HUIGE, CJM ;
ALTONA, C .
JOURNAL OF COMPUTATIONAL CHEMISTRY, 1995, 16 (01) :56-79
[8]  
Kolodny E., 1995, METABOLIC MOL BASES, P2693
[9]   Regulated diversity of heparan sulfate [J].
Lindahl, U ;
Kusche-Gullberg, M ;
Kjellén, L .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (39) :24979-24982
[10]   Crystal structure of human arylsulfatase A: The aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis [J].
Lukatela, G ;
Krauss, N ;
Theis, K ;
Selmer, T ;
Gieselmann, V ;
von Figura, K ;
Saenger, W .
BIOCHEMISTRY, 1998, 37 (11) :3654-3664