Location: Bioenergy Research Unit
Title: Activation of a GH43 ß-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity Authors
Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 27, 2013
Publication Date: March 13, 2013
Citation: Jordan, D.B., Lee, C.C., Wagschal, K., Braker, J.D. 2013. Activation of a GH43 ß-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity. Archives of Biochemistry and Biophysics. 533:79-87. Interpretive Summary: Conversion of plant polymers to simple sugars for subsequent fermentation to biofuels and other products can be achieved most cost efficiently by enzyme-catalyzed saccharification. This applies to hemicellulose, the second most abundant plant polymer after cellulose. This work examines the newly discovered ß-xylosidase of GH43 family of enzymes that is strongly activated by divalent metal cations. ß-Xylosidase are responsible for the final step in the breakdown of hemicellulose, releasing the simple sugar xylose that can then be fermented. Here we demonstrate that the activation by divalent metal can be slow or rapid depending on the metal. We also report the steady-state kinetic parameters of the enzyme activated by each metal activator. The enzyme exhibits the highest kcat/Km of any enzyme in catalyzing the hydrolysis of xylooligosaccharides.
Technical Abstract: RS223-BX of glycoside hydrolase family 43 is a ß-xylosidase that is strongly activated (kcat/Km as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+and Ni2+. Slow activation by Mg2+ was demonstrated (kon 0.0130 s-1 mM-1, koff 0.00789 s-1) at pH 7.0 and 25 °C. Several-fold faster values of koff and kon were estimated for Ca2+. koff and kon values are independent of Mg2+ concentration, but koff and kon are slower in the presence of increasing levels of substrate 4-nitrophenyl ß-D-xylopyranoside (4NPX). Binding kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E·M2+, followed by slow isomerization to the activated enzyme, E*·M2+. In addition, the slower kon and koff values in the presence of 4NPX strongly suggest that M2+ binds productively to the enzyme active site before 4NPX. Steady-state kinetic parameters of each divalent metal activated RS223-BX acting on natural and artificial substrates were determined. Moderately high values of kcat (7 to 30 s-1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25 °C. Certain M2+-activated RS223-BX exhibit the highest reported values of kcat/Km of any ß-xylosidase acting on natural substrates: for example, at pH 7.0 and 25 °C, xylobiose (Mn2+, 190 mM-1s-1), xylotriose (Ca2+, 150 mM-1s-1) and xylotetraose (Ca2+, 260 mM-1s-1). There is potential of the enzyme to add value to industrial saccharification operations at low substrate and high D-glucose and D-xylose concentrations.