Phospholipase A2 engineering. 3. Replacement of lysine-56 by neutral residues improves catalytic efficiency significantly, alters substrate specificity, and clarifies the mechanism of interfacial recognition

1990 ◽  
Vol 112 (9) ◽  
pp. 3704-3706 ◽  
Author(s):  
Joseph P. Noel ◽  
Tiliang Deng ◽  
Kelly J. Hamilton ◽  
Ming Daw Tsai
Keyword(s):  
Substrate Specificity ◽  
Phospholipase A2 ◽  
Catalytic Efficiency

scholarly journals A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

2020 ◽  
Vol 295 (51) ◽  
pp. 17752-17769
Author(s):  
Evan M. Glasgow ◽  
Elias I. Kemna ◽  
Craig A. Bingman ◽  
Nicole Ing ◽  
Kai Deng ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Design ◽  
Plant Biomass ◽  
Catalytic Efficiency ◽  
Structural Features ◽  
Glycoside Hydrolases ◽  
Design Strategies ◽  
Biomass Hydrolysis ◽  
Diverse Range ◽  
Broad Specificity

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)–linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

scholarly journals WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

2005 ◽  
Vol 187 (20) ◽  
pp. 7056-7061 ◽  
Author(s):  
Loes E. Bevers ◽  
Emile Bol ◽  
Peter-Leon Hagedoorn ◽  
Wilfred R. Hagen
Keyword(s):  
Substrate Specificity ◽  
Pyrococcus Furiosus ◽  
Catalytic Efficiency ◽  
Aromatic Aldehydes ◽  
Protein Subunit ◽  
Efficient Catalyst ◽  
Paramagnetic Resonance ◽  
Broad Substrate Specificity ◽  
Aldehyde Oxidoreductase ◽  
Redox Partner

ABSTRACT WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V max = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus.

Kinetic Basis for the Substrate Specificity during Hydrolysis of Phospholipids by Secreted Phospholipase A2†

Biochemistry ◽  
1996 ◽  
Vol 35 (29) ◽  
pp. 9375-9384 ◽  
Author(s):  
Joseph Rogers ◽  
Bao-Zhu Yu ◽  
Spyros V. Serves ◽  
Gerasimos M. Tsivgoulis ◽  
Demetrios N. Sotiropoulos ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Phospholipase A2 ◽  
Secreted Phospholipase A2 ◽  
Hydrolysis Of

scholarly journals Functionalized Bilayer Membranes as Artificial Tryptophan Synthase. Characterization of Catalytic Efficiency, Substrate Specificity, and Reaction Selectivity

1990 ◽  
Vol 63 (8) ◽  
pp. 2339-2345 ◽  
Author(s):  
Yukito Murakami ◽  
Jun-ichi Kikuchi ◽  
Yoshio Hisaeda ◽  
Koichiro Nakamura ◽  
Tomoyuki Kitazaki ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Efficiency ◽  
Tryptophan Synthase ◽  
Bilayer Membranes ◽  
Reaction Selectivity

Presence of Glycerol Masks the Effects of Phosphorylation on the Catalytic Efficiency of Cytosolic Phospholipase A2

1997 ◽  
Vol 341 (1) ◽  
pp. 177-185 ◽  
Author(s):  
James R. Burke ◽  
Matthew G. Guenther ◽  
Mark R. Witmer ◽  
Jeffrey A. Tredup ◽  
Mark E. Hail ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Catalytic Efficiency ◽  
Cytosolic Phospholipase A2 ◽  
Cytosolic Phospholipase

scholarly journals Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

2003 ◽  
Vol 69 (8) ◽  
pp. 4438-4447 ◽  
Author(s):  
Jung-Kul Lee ◽  
Bong-Seong Koo ◽  
Sang-Yong Kim ◽  
Hyung-Hwan Hyun
Keyword(s):  
Substrate Specificity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Mannitol Dehydrogenase ◽  
High Substrate ◽  
Candida Magnoliae

ABSTRACT Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k cat = 823 s−1, K m = 28.0 mM, and k cat /K m = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

scholarly journals Characterization of phospholipase A2 in monocytic cell lines. Functional and biochemical aspects of membrane association

1991 ◽  
Vol 276 (3) ◽  
pp. 631-636 ◽  
Author(s):  
W Rehfeldt ◽  
R Hass ◽  
M Goppelt-Struebe
Keyword(s):  
Substrate Specificity ◽  
Phospholipase A2 ◽  
Cell Lines ◽  
Cytosolic Phospholipase A2 ◽  
Biologically Active ◽  
Ph Optimum ◽  
Monocytic Cell ◽  
Cytosolic Phospholipase ◽  
Membrane Bound ◽  
Phospholipase A2 Activity

Phospholipase A2 activity was characterized in the human monocytic tumour-cell lines U937 and THP1. The enzyme showed an alkaline pH optimum and substrate specificity for arachidonoyl-phosphatidylcholine. The activation of phospholipase A2 required bivalent cations (Ca2+ greater than Mg2+ = Sr2+ greater than Ba2+). Investigation of the subcellular distribution of the enzyme revealed that the phospholipase A2 activity was shifted to the cytosol in the presence of EDTA, indicating that the association of the enzyme with the cellular membranes is Ca2+ (bivalent-cation)-dependent. Stimulation of THP1 cells for 2-4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) activated cytosolic and membrane-bound phospholipase A2. At this time, no effect of PMA on phospholipase A2 activity was observed in the less mature U937 cells. However, when both cell lines were induced to differentiate along the monocytic pathway by a 2-3-day treatment with PMA, the cells released significant amounts of arachidonic acid and prostanoids. Compared with undifferentiated control cells, these PMA-differentiated cells showed a decrease in cytosolic phospholipase A2 activity and an increase in membrane-bound activity. Membrane-bound and cytosolic enzyme showed the same pH optimum, Ca(2+)-dependency and substrate specificity. These data indicate that membrane-bound and cytosolic phospholipase A2 activities represent one enzyme and that the membrane-bound form is the biologically active phospholipase A2.

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