The substrate-specificity of human lysosomal α-d-mannosidase in relation to genetic α-mannosidosis

The specificity of human liver lysosomal alpha-mannosidase (EC 3.2.1.24) towards a series of oligosaccharide substrates derived from high-mannose, complex and hybrid asparagine-linked glycans and from the storage products in alpha-mannosidosis was investigated. The enzyme hydrolyses all alpha(1-2)-, alpha(1-3)- and alpha(1-6)-mannosidic linkages in these glycans without a requirement for added Zn2+, albeit at different rates. A major finding of this study is that all the substrates are hydrolysed by non-random pathways. These pathways were established by determining the structures of intermediates in the digestion mixtures by a combination of h.p.t.l.c. and h.p.l.c. before and after acetolysis. The catabolic pathway for a particular substrate appears to be determined by its structure, raising the possibility that degradation occurs by an uninterrupted sequence of steps within one active site. The structures of the digestion intermediates are compared with the published structures of the storage products in mannosidosis and of intact asparagine-linked glycans. Most but not all of the digestion intermediates derived from high-mannose glycans have structures found in intact asparagine-linked glycans of human glycoproteins or among the storage products in the urine of patients with mannosidosis. However, the relative abundances of these structures suggests that the catabolic pathway is not the same as the processing pathway. In contrast, the intermediates formed from the digestion of oligosaccharides derived from hybrid and complex N-glycans are completely different from any processing intermediates and also from the oligosaccharides of composition Man2-4GlcNAc that account for 80-90% of the storage products in alpha-mannosidosis. It is postulated that the structures of these major storage products arise from the action of an exo/endo-alpha(1-6)-mannosidase on the partially catabolized oligomannosides that accumulate in the absence of the main lysosomal alpha-mannosidase.

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Substrate specificity of human liver neutral α-mannosidase

Biochemical Journal ◽

10.1042/bj2860047 ◽

1992 ◽

Vol 286 (1) ◽

pp. 47-53 ◽

Cited By ~ 10

Author(s):

S al Daher ◽

R De Gasperi ◽

P Daniel ◽

S Hirani ◽

C Warren ◽

...

Keyword(s):

Substrate Specificity ◽

Human Liver ◽

Core Structure ◽

Catabolic Pathways ◽

Mannose Residue ◽

The Core ◽

High Mannose

The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol.

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Locating Al in the DABCO catalyst before and after Al extraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100174461 ◽

1990 ◽

Vol 48 (4) ◽

pp. 265-267

Author(s):

Kathleen B. Reuter

Keyword(s):

Active Site ◽

Inverse Relationship ◽

Transverse Direction ◽

Reaction Rate ◽

Acid Phosphate ◽

Fracture Surfaces ◽

Al Content ◽

Before And After ◽

Relationship Of

The reaction rate and efficiency of piperazine to 1,4-diazabicyclo-octane (DABCO) depends on the Si/Al ratio of the MFI topology catalysts. The Al was shown to be the active site, however, in the Si/Al range of 30-200 the reaction rate increases as the Si/Al ratio increases. The objective of this work was to determine the location and concentration of Al to explain this inverse relationship of Al content with reaction rate.Two silicalite catalysts in the form of 1/16 inch SiO2/Al2O3 bonded extrudates were examined: catalyst A with a Si/Al of 83; and catalyst B, the acid/phosphate Al extracted form of catalyst A, with a Si/Al of 175. Five extrudates from each catalyst were fractured in the transverse direction and particles were obtained from the fracture surfaces near the center of the extrudate diameter. Particles were also obtained from the outside surfaces of five extrudates.

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The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657715 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1202-1208 ◽

Cited By ~ 18

Author(s):

Marianne Kjalke ◽

Julie A Oliver ◽

Dougald M Monroe ◽

Maureane Hoffman ◽

Mirella Ezban ◽

...

Keyword(s):

Tissue Factor ◽

Active Site ◽

Large Scale ◽

Thrombin Generation ◽

Factor Viia ◽

Dependent Manner ◽

Antithrombotic Agent ◽

Before And After ◽

Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

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A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Archives of Biochemistry and Biophysics ◽

10.1016/s0003-9861(04)00230-9 ◽

2004 ◽

Author(s):

J RAMAN

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Site Mutation ◽

Hypoxanthine Guanine Phosphoribosyltransferase

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Substrate Specificity and Ligand Interactions of CYP26A1, the Human Liver Retinoic Acid Hydroxylase

Molecular Pharmacology ◽

10.1124/mol.111.072413 ◽

2011 ◽

Vol 80 (2) ◽

pp. 228-239 ◽

Cited By ~ 23

Author(s):

Jayne E. Thatcher ◽

Brian Buttrick ◽

Scott A. Shaffer ◽

Jakob A. Shimshoni ◽

David R. Goodlett ◽

...

Keyword(s):

Retinoic Acid ◽

Substrate Specificity ◽

Human Liver ◽

Ligand Interactions

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Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

Journal of Biological Chemistry ◽

10.1074/jbc.274.4.2344 ◽

1999 ◽

Vol 274 (4) ◽

pp. 2344-2349 ◽

Cited By ~ 125

Author(s):

Shinya Oue ◽

Akihiro Okamoto ◽

Takato Yano ◽

Hiroyuki Kagamiyama

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Cumulative Effects ◽

Active Site Residues

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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