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Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity

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Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity Structure 14, 567–575, March 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.str.2005.11.016 Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity Andreas H. Ehrensberger,1,2,3 Robert A. Elling,1,2 is catalyzed by xylose reductase (XR), an NADPH-pre- and David K. Wilson1,2,* ferring enzyme which reduces xylose to form xylitol (Fig- 1 Section of Molecular and Cellular Biology ure 1). Xylitol dehydrogenase (XDH) reoxidizes xylitol University of California, Davis to form xylulose, using NAD+ exclusively as a cosub- Davis, California 95616 strate. Finally, xylulose kinase phosphorylates xylulose at the 5 position using ATP. The xylose-to-xylulose con- version can also be catalyzed in one step by xylose Summary isomerase, a prokaryotic enzyme. Attempts to express various forms of this enzyme in yeast have been largely Xylitol dehydrogenase (XDH) is one of several en- unsuccessful, with one recent exception (Kuyper et al., zymes responsible for assimilating xylose into eukary- 2005). otic metabolism and is useful for fermentation of xy- One possible solution to the cosubstrate recycling lose contained in agricultural byproducts to produce problem can be obtained with an NADH-dependent ethanol. For efficient xylose utilization at high flux XR. Progress in this area has been made by searching rates, cosubstrates should be recycled between the for enzymes with a rare natural preference for this co- NAD+-specific XDH and the NADPH-preferring xylose substrate. The XR from Candida parapsilosis possesses reductase, another enzyme in the pathway. To under- an approximately 100-fold preference for NADH over stand and alter the cosubstrate specificity of XDH, NADPH based on the ratio of catalytic efficiencies, we determined the crystal structure of the Glucono- kcat/Km (Lee et al., 2003). Alternatively, the abundance bacter oxydans holoenzyme to 1.9 A˚ resolution. The of biochemical and structural knowledge of the XR structure reveals that NAD+ specificity is largely con- from Candida tenuis has been exploited to design mu- ferred by Asp38, which interacts with the hydroxyls tants with altered cosubstrate specificity. Currently, of the adenosine ribose. Met39 stacked under the pu- the wild-type enzyme, which has a 33-fold preference rine ring and was also located near the 20 hydroxyl. for NADPH, has been altered to exhibit a 5-fold prefer- Based on the location of these residues and on se- ence for NADH (Petschacher et al., 2005). quence alignments with related enzymes of various As an alternative approach to alleviate the cosub- cosubstrate specificities, we constructed a double strate recycling problem, the cosubstrate specificity of mutant (D38S/M39R) that was able to exclusively use XDH can be changed to NADP+. Xylitol dehydrogenase, NADP+, with no loss of activity. also known as D-xylulose reductase, from Glucono- bacter oxydans (E.C. 1.1.1.9) is among the best-studied XDHs and has been shown to exclusively use NAD+.No Introduction structural data are, however, available for this or any other XDH. Based on its sequence, G. oxydans XDH The metabolic integration of xylose, the second most has been classified as a member of the superfamily of abundant sugar in nature after glucose, by yeast could short chain dehydrogenases/reductases (SDR, also play a key role in the bioconversion of lignocellulose- known as the short chain oxidoreductase/SCOR family), containing agricultural waste products to ethanol via which currently encompasses approximately 3000 pro- fermentation. A majority of postharvest residues are cur- teins. As a group, SDRs can accommodate a wide range rently disposed of through burning, a practice that pro- of substrate sizes and polarities and are found in all spe- duces greenhouse gases and leads to air pollution and cies. Enzymes with XDH activity also exist in the diver- associated human health problems. These waste prod- gent medium chain dehydrogenase/reductase (MDR) ucts could serve as an energy source if converted to eth- superfamily and cosubstrate engineering experiments anol, which has the potential to drive hydrogen fuel cells converting the cosubstrate specificity have been carried (Deluga et al., 2004). Although many five-carbon sugars out on a form from Pichia stipitis (Watanabe et al., 2004). are utilized by Saccharomyces cerevisiae, an organism In addition to its ability to oxidize xylitol, G. oxydans XDH that has been optimized for ethanol production, xylose has previously been shown to use sorbitol and mannitol is not. Efficient fermentation of xylose in budding yeast as relatively good substrates (Sugiyama et al., 2003). is not possible because the organism lacks the enzymes SDRs are characterized by a set of recurring se- necessary to integrate this sugar into its metabolism. quence motifs (Oppermann et al., 2003) and by a cata- Previous attempts have been made to express the en- lytic Ser-Tyr-Lys-Asn tetrad. Most are oxidoreductases zymes necessary for conversion of xylose into xylu- that utilize NAD+ or NADP+, and of the over 40 structures lose-5-phosphate, which can enter metabolism via the determined so far, all contain a single Rossmann fold pentose phosphate pathway (most recently reviewed domain. In many cases, SDRs assemble into dimers or by Jeffries and Jin, 2004). The first of these reactions tetramers, although monomers have been found to exist as well (Ghosh et al., 2001; Oppermann et al., 2003). As a result of these structural studies, the atomic interac- *Correspondence: [email protected] tions determining cosubstrate specificity are under- 2 Lab address: http://alanine.ucdavis.edu/wdave/wilsonlab/index. html stood and it has been possible to make relatively accu- 3 Present address: Biophysics Program, Stanford University, Stan- rate predictions regarding this selectivity (Duax et al., ford, California 94305. 2003; Persson et al., 2003). Structure 568 Figure 1. A Schematic of the Eukaryotic Xy- lose Assimilation Pathway The crystal structure of NAD+-bound XDH has been NAD+ Binding determined to a resolution of 1.9 A˚ with an R factor of The axis of the NAD+ runs roughly parallel to the b sheet 16.7%. The high-resolution structure reveals details and perpendicular to the six Rossmann fold helices. The about catalysis and substrate and cosubstrate specific- N-terminal region of the primary sequence is responsi- ity, and allowed us to design a double mutant specific ble for binding the adenine portion of the cofactor and for NADP+ utilization. By introducing two mutations establishing selectivity for NAD+ or NADP+. The nicotin- into XDH, we have developed an enzyme that may allow amide ring which defines the active site of the enzyme is S. cerevisiae cells to metabolize xylose without the confined between helices aFG1 and aFG2 and the C-ter- problem of cosubstrate depletion. minal side of the b sheet. The NAD+ is anchored to the enzyme with the nicotinamide ring in the syn conforma- tion, which is stabilized in this conformation by an intra- Results and Discussion molecular hydrogen bond between the nicotinamide amide and the pyrophosphate (Figure 3). Both ribose hy- Overall Structure droxyls engage in hydrogen bonds with Lys161, and the 20 hydroxyl forms a hydrogen bond with the catalytic G. oxydans XDH crystallized in space group P21 with two tetramers in the asymmetric unit. Phasing was carried residue; Tyr157 has been shown to form part of the pro- out via molecular replacement using the 38% identical ton conduction system to and from the active site (Filling Corynebacterium aquaticum levodione reductase tetra- et al., 2002). mer (Sogabe et al., 2003) as the search model. Each tet- The adenine ribose is positioned in anti conformation, ramer is assembled via noncrystallographic 222 symme- with the adenine ring stacking adjacent to Met39. Both try and contains one clearly observable NAD+ molecule ribose hydroxyls form hydrogen bonds with Asp38, the + in each of its monomers (Figure 2A). The tetramer re- key polar determinant of NAD specificity. The adenine sembles a flat torus and has an approximate diameter primary amine forms a hydrogen bond with Asp64. 0 of 80 A˚ . Of the 262 residues constituting each of the in- Both riboses on the molecule adopt the C2 -endo con- tact monomers (designated A–H), residues 3–262 were formation. The pyrophosphate moiety interacts primar- fit into the electron density. There is very little structural ily with water molecules and contacts the protein only divergence between the monomers, with the rms devia- through Asn18 and the backbone amide nitrogen of tion between them ranging from 0.18 to 0.28 A˚ . All refer- Ile19. ences to a particular feature of the XDH structure will in this paper refer to monomer E, as it subjectively ex- Magnesium and Metal Binding to XDH hibited the best-defined electron density. Five small spherical regions of high electron density Interactions between monomers within the tetramer (5 to 6 s in the 2Fo 2 Fc map) were found in the asymmet- are stabilized on two sides by hydrogen bonds between ric unit. Because the crystallization buffer contained adjacent 7-stranded b sheets, forming two 14-strand 100 mM MgCl2, and the spheres exhibited octahedral ge- wide parallel sheets. The other two sides of the tetramer ometry typical for divalent cations, five magnesium ions are stabilized by four-helix bundle interactions between were assigned to these densities. All five ions are located two helices on one monomer and two helices on the far from the active sites and substrate binding pockets neighboring monomer (Figure 2B). and are therefore likely to have no direct impact on catal- Each monomer contains a single Rossmann fold do- ysis. The shortest distance from the C4 of a nicotinamide main consisting of a large 7-stranded b sheet flanked to a magnesium is 16 A˚ . Two Mg2+ ions lie directly on by three short helices on one side (aB, aC, and aG, using a noncrystallographic 2-fold axis (the horizontal axis in conventional SDR nomenclature) and three longer heli- Figure 2A) between two monomers in similar environ- ces (aD, aE, and aF) on the other side of the sheet ments, while the fifth ion lies outside both tetramers.
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