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. 2005 May;17(5):1598-611.
doi: 10.1105/tpc.104.029983. Epub 2005 Apr 13.

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase

Erin K Bomati  1 , Joseph P Noel
Affiliations

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase

Erin K Bomati et al. Plant Cell. 2005 May.

Abstract

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

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Figures

Figure 1.
Figure 1.
Biosynthesis of Monolignols. (A) Monolignols. (B) Monolignol biosynthesis. p-Coumaryl alcohol: R1 = R2 = H; coniferyl alcohol: R1 = OCH3, R2 = H; sinapyl alcohol: R1 = R2 = OCH3.
Figure 2.
Figure 2.
Three-Dimensional Architecture of the Aspen SAD/NADP+ Complex. (A) Ribbon representation of the aspen SAD dimer. Monomer A is colored blue, monomer B is colored rose, and NADP+ is bound in the nucleotide binding domain. The active site cavity is boxed in orange. Each monomer coordinates one structural and one catalytic Zn2+ ion, each represented as green spheres with small black spheres indicating coordination bonds. (B) 2Fo-Fc electron density map of NADP+ contoured at 1.1 σ. (C) Hydrogen bond formation (small green spheres) establishes a proton shuttle. (D) Ball-and-stick representation of NADP+ phosphate-stabilizing residues.
Figure 3.
Figure 3.
The Aspen SAD Active Site. SAD active site architecture is defined by residues from both monomers. The catalytic Zn2+ ion is tetrahedrally coordinated by Cys-50, His-72, Cys-166, and a water molecule, represented as a red sphere. Small green spheres indicate a hydrogen bond formation between the Zn2+ ion coordinating water and Ser-52. A ball-and-stick model of NADP+ is shown. The site of hydride transfer is labeled with an asterisk.
Figure 4.
Figure 4.
The SAD Substrate Binding Pocket. (A) and (C) Docking solutions for sinapaldehyde and coniferaldehyde (shown with black carbon atoms), respectively. The phenyl rings pack flat against the base of the active site, with the aldehyde carbonyls of the substrates directly coordinating the Zn2+ ion. (B) and (D) Surface representation of the SAD active site cavity. The surface corresponding to Gly-302 is shaded yellow.
Figure 5.
Figure 5.
Partial Sequence Alignment of SAD-Like and CAD-Like Enzymes. Alignment of key residues in SAD-like (Populus tremuloides SAD [PtSAD], Fragaria ananassa CAD1 [FxaCAD1], Arabidopsis thaliana BAD/ELI3-2 [AtBAD], A. thaliana CAD6 [AtCAD6], and Petroselinum crispum CAD [PcELI3]) and classical CAD-like (P. tremuloides CAD [PtCAD], Eucalyptus gunnii CAD2 [EuCAD2], A. thaliana CAD4 [AtCAD4], A. thaliana CAD5 [AtCAD5], and Pinus taeda CAD [Pt*CAD]) enzymes. Catalytic Zn2+ ion coordinating residues are shaded blue; proton shuttle residues are shaded orange; key bulky active site residues are shaded purple; structural Zn2+ ion coordinating residues are shaded green; residues defining active site topology are shaded pink; and the residue we propose to be the key determinant of substrate specificity is shaded yellow.
Figure 6.
Figure 6.
Substrate Docking in the Modeled Active Sites of SAD/CAD Family Enzymes. (A) Docking solution for sinapaldehyde (shown with black carbon atoms) in the FxaCAD active site. The phenyl ring packs flat against the base of the active site in the same orientation as for sinapaldehyde docked in the SAD active site. (B) Surface representation of the modeled FxaCAD active site cavity. The surface corresponding to Gly-300 is shaded yellow. (C) Docking solutions for coniferaldehyde (shown with black carbon atoms) in the PcELI3 active site. Because of the bulk of the Asn side chain, the phenyl ring packs on its unsubstituted edge against the base of the active site. (D) Surface representation of the modeled PcELI3 active site cavity. The surface corresponding to Asn-280 is shaded yellow. (E) Docking solutions for 2-methoxybenzaldehyde (shown with black carbon atoms) in the AtBAD active site. Because of the bulk of the Met side chain, the active site is largely occluded. (F) Surface representation of the modeled AtBAD active site cavity. The surface corresponding to Met-298 is shaded yellow.
Figure 7.
Figure 7.
Initial Velocity versus Substrate Concentration Plots of Wild-Type SAD-Catalyzed Reactions. (A) Wild-type SAD-catalyzed reduction of sinapaldehyde at low substrate concentrations. The inset shows substrate inhibition occurring at concentrations exceeding 60 μM. (B) Wild-type SAD-catalyzed reduction of coniferaldehyde at low substrate concentrations. The inset shows substrate inhibition occurring at concentrations exceeding 100 μM.
Figure 8.
Figure 8.
Initial Velocity versus Substrate Concentration Plots of Mutant SAD-Catalyzed Reactions. (A) F289P/W61L SAD-catalyzed reduction of sinapaldehyde. (B) F289P/W61L SAD-catalyzed reduction of coniferaldehyde at low substrate concentrations. The inset shows moderate substrate inhibition occurring at coniferaldehyde concentrations exceeding 2.0 mM. (C) F289P/W61L/G302F/L122W SAD-catalyzed reduction of sinapaldehyde. (D) F289P/W61L/G302F/L122W SAD-catalyzed reduction of coniferaldehyde at low substrate concentrations. The inset shows substrate inhibition occurring at coniferaldehyde concentrations exceeding 1.75 mM. (E) G302F/L122W SAD-catalyzed reduction of sinapaldehyde. (F) G302F/L122W SAD-catalyzed reduction of coniferaldehyde at low substrate concentrations. The inset shows substrate inhibition occurring at coniferaldehyde concentrations exceeding 90 μM.
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