Caroline S.M. Furniss

The Dual Nature of the Wheat Xylanase Protein Inhibitor XIP-I: STRUCTURAL BASIS FOR THE INHIBITION OF FAMILY 10 AND FAMILY 11 XYLANASES.

The xylanase inhibitor protein I (XIP-I) from wheat Triticum aestivum is the prototype of a novel class of cereal protein inhibitors that inhibit fungal xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). The crystal structures of XIP-I in complex with Aspergillus nidulans (GH10) and Penicillium funiculosum (GH11) xylanases have been solved at 1.7 and 2.5 Å resolution, respectively. The inhibition strategy is novel because XIP-I possesses two independent enzyme-binding sites, allowing binding to two glycoside hydrolases that display a different fold. Inhibition of the GH11 xylanase is mediated by the insertion of an XIP-I Π-shaped loop (Lα4β5) into the enzyme active site, whereas residues in the helix α7 of XIP-I, pointing into the four central active site subsites, are mainly responsible for the reversible inactivation of GH10 xylanases. The XIP-I strategy for inhibition of xylanases involves substrate-mimetic contacts and interactions occluding the active site. The structural determinants of XIP-I specificity demonstrate that the inhibitor is able to interact with GH10 and GH11 xylanases of both fungal and bacterial origin. The biological role of the xylanase inhibitors is discussed in light of the present structural data.

Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. The K(i) values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-10 and -11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I-A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of -1.38 kJ x mol(-1) x K(-1), leading to a predicted buried surface area of approx. 2200+/-500 A(2) at the complex interface. For this complex with A. niger xylanase (K(i)=320 nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4-7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (K(i)=9 nM) led to an observable interaction across the entire pH range tested (3-9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate k(off) for the latter, with only a small difference in association rate k(on).

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.

Comparison of modular and non-modular xylanases as carrier proteins for the efficient secretion of heterologous proteins from Penicillium funiculosum

Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domains separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a host for the secreted production of heterologous proteins, each of the xylanases was assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the β-glucuronidase (GUS) protein from Escherichia coli is secreted by P. funiculosum when expressed as an XYNA fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycosylated and enzymatically inactive.

Expression of a Brassica isopropylmalate synthase gene in Arabidopsis perturbs both glucosinolate and amino acid metabolism.

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants.

THE MANIPULATION OF DNA WITH RESTRICTION ENZYMES IN LOW WATER SYSTEMS

The cleavage of phage lambda (lambda) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied--those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII.