Abstract
Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40(T), SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C(+) domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C(+) in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.
Highlights
Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains
Whereas two family GH4 ␣-glucuronidases from Thermotoga maritime (TM0434 and TM0752) hydrolyze p-nitrophenyl-␣-D-glucuronopyranoside, both lack activity toward 4-Omethyl-D-glucuronoxylan [13, 14]; TM0752 lacks activity toward aldouronic acids [13], whereas TM0434 activity toward aldouronic acids has not been tested
A Five-domain GH115 ␣-Glucuronidase characterized ␣-glucuronidases from family GH115, on the other hand, are able to remove (Me)GlcAp from both terminal and internal Xylp units in xylan and xylooligomers [16, 17, 22, 23] and so may be beneficial to efforts aimed at harnessing xylan as a polymer
Summary
Residual activity was measured by transferring 6.0 g of the heat-treated enzyme to a final 0.2-ml reaction mixture comprising 200 mM MES (pH 6.5) and 1.0% (w/v) beechwood xylan. SdeAgu115A was concentrated to 13.0 mg mlϪ1 in 10 mM HEPES (pH 7.5) containing 500 mM NaCl. The crystallization screen was performed by mixing 0.5 l of protein with 0.5 l of reservoir buffer using a Mosquito crystal (TTPlab) liquid-handling system. The SdeAgu115A structure was determined by the singlewavelength anomalous diffraction method using phasing, density modification, and initial protein model building as implemented in the HKL3000 software package [45,46,47,48,49,50,51]. Enzyme dose was adjusted to measure initial rates of reaction at 40 °C over 10 min (0.1, 0.7, 0.7, and 140 g of SdeAgu115A for beechwood xylan, U4m2XX, XU4m2XX, and pNP-GlcAp, respectively). Electrospray ionization mass spectrometric analyses were performed using a Q-Exactive mass spectrometer (Thermo Scientific) in negative mode, and product spectra were compared with D-glucuronic acid
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