Structural and Kinetic Basis for Substrate Selectivity in <i>Populus tremuloides</i> Sinapyl Alcohol Dehydrogenase

Bomati, E.K.; Noel, Joseph P.

doi:10.1105/tpc.104.029983

Cited by 74 publications

(94 citation statements)

References 32 publications

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“…2D), from Populus tremuloides (aspen), was described in 2005 [7]. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde, as well as substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes.…”

Section: Lignin Lignans and Phenylpropenesmentioning

confidence: 53%

“…These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. The SAD three-dimensional structure was used to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, it was possible to draw correlations between substrate specificity and amino acid substitution patterns in their active sites [7].…”

Section: Lignin Lignans and Phenylpropenesmentioning

confidence: 99%

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Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

Ferrer

Austin

Stewart

et al. 2008

Plant Physiology and Biochemistry

700

413

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As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4′,5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure-function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and ongoing evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.

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Section: Lignin Lignans and Phenylpropenesmentioning

confidence: 53%

Section: Lignin Lignans and Phenylpropenesmentioning

confidence: 99%

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

Ferrer

Austin

Stewart

et al. 2008

Plant Physiology and Biochemistry

700

413

View full text Add to dashboard Cite

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“…However, the removal of (or a decrease in) substrate inhibition is generally accompanied by reduced catalytic activity in many enzymes including lactate dehydrogenase, 17␤-hydroxysteroid dehydrogenase, D-phosphoglycerate dehydrogenase, sinapyl alcohol dehydrogenase, and tetrahydroquinone dehalogenase (39,(42)(43)(44)(45). Compared with wild-type SalR, the independent F104A and I275A mutants displayed weak substrate inhibition accompanied by similar or even increased reaction velocities.…”

Section: Discussionmentioning

confidence: 99%

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Ziegler

Brandt

Geißler

et al. 2009

Journal of Biological Chemistry

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Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel ␣-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.

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“…Structures were obtained from the RCSB Protein Data Bank: Coniferaldehyde and sinapaldehyde from Louie et al (2010), and NADP + from Bomati and Noel (2005) or Youn et al (2006). We conducted flexible ligand docking of NADP + to the CtCADs, which was followed with flexible ligand docking of substrates (coniferaldehyde and sinapaldehyde) to the CtCAD-NADP + complex, cycling through 1,000 maximum orientations for each docking procedure.…”

Section: Molecular Dockingmentioning

confidence: 99%

“…The SWISS-MODEL automated protein structure homologymodeling server (Arnold et al 2006;Bordoli et al 2009) used the PtreSAD crystal structure (PDB ID: 1YQD) (Bomati and Noel 2005) as the template for CtCAD1 and CtCAD3, and AtCAD5 crystal structure (PDB ID: 2CF6) (Youn et al 2006) as the template for CtCAD2. Identity between the CtCADs and their respective templates ranged between 68 to 79%.…”

Section: Homology Modeling and Molecular Dynamicsmentioning

confidence: 99%

Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius

Ragamustari

Shiraiwa

Hattori

et al. 2013

Plant Biotechnology

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We report the isolation and characterization of three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius (safflower). All three recombinant CADs were able to reduce coniferaldehyde and sinapaldehyde into coniferyl alcohol and sinapyl alcohol, respectively, and were designated as CtCAD1, CtCAD2, and CtCAD3. Phylogenetic analysis of CAD amino acid sequences and homology modeling revealed that CtCAD1 and CtCAD3 were closely related to the sinapaldehyde-specific aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (PtreSAD). CtCAD2 was in a clade containing class I plant CADs. Gas chromatography-mass spectrometry-based kinetic analysis using two different substrates, coniferaldehyde and sinapaldehyde, indicated that the CtCADs showed no strong preference for either substrate. CtCAD2 has the highest catalytic efficiency (k cat /K m ) (81.49 mM −1 min −1 and 95.3 mM −1 min −1 for coniferaldehyde and sinapaldehyde, respectively) compared with the other CtCADs. Inhibition kinetics showed that coniferaldehyde was a stronger inhibitor than sinapaldehyde for all CtCADs. Quantitative real-time PCR revealed that CtCAD2 was expressed at higher levels than CtCAD1 and CtCAD3 in all samples, except developing seeds at 3 days after flowering, where CtCAD1 had a higher expression level. In plant protein assays with coniferaldehyde and sinapaldehyde, plant protein extracted from seeds at 7 days after flowering, showed the highest specific activity. The product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 and CtCAD3 in the respective organs.

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Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

Cited by 74 publications

References 32 publications

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius

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