Apo and Holo Structures of an NADP(H)-dependent Cinnamyl Alcohol Dehydrogenase from Saccharomyces cerevisiae

Valencia, Eva; Larroy, Carol; Ochoa, Wendy F.; Parés, Xavier; Fita, Ignacio; Biosca, Josep A.

doi:10.1016/j.jmb.2004.06.037

Cited by 51 publications

(48 citation statements)

References 50 publications

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“…The predominant form of CAD is both NADPH and Zn 2+ dependent and belongs to the medium-chain dehydrogenase/ reductase (MDR) superfamily. The crystal structure of Arabidopsis CAD5 has been reported (Youn et al, 2006) and is similar to the structure of an aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (Bomati and Noel, 2005) and a putative CAD from yeast (Saccharomyces cerevisiae) (Valencia et al, 2004). Both Arabidopsis CAD5 and aspen sinapyl alcohol dehydrogenase are homodimeric, with monomeric subunits each carrying a tetrahedrally coordinated catalytic zinc ion in addition to a second structural zinc ion.…”

Section: Introductionmentioning

confidence: 69%

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis

et al. 2014

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The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/ reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4-to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.

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Section: Introductionmentioning

confidence: 69%

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis

et al. 2014

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“…As a means of comparison, MCADH can reduce a broad range of aldehydes, including propanal, butanal, hexanal, octanal, and benzaldehyde (9,17,20). Studies conducted with the MCADH enzyme from Saccharomyces cerevisiae did not test the activity of the enzyme with longer-chain substrates such as decanal and dodecanal.…”

Section: Resultsmentioning

confidence: 99%

Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8

Wahlen

Oswald

Seefeldt

et al. 2009

Appl Environ Microbiol

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Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP ؉ . The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.

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“…This correlation will be of great value for understanding and possibly predicting the substrate specificities of uncharacterized SAD-like enzymes and for providing a structure platform to begin the rational engineering of new specificities in this family of enzymes. Although SAD bears only 36% sequence identity to the recently characterized Saccharomyces cerevisiae CAD (ScCAD), the three-dimensional structures of these two enzymes share a similar backbone fold, with the alignment of Ca backbone atoms overlaying with an RMSD of 1.55 Å across 335 residues (Valencia et al, 2004). Unlike SAD, ScCAD does not reduce sinapaldehyde or coniferaldehyde, selectively using cinnamaldehyde as a substrate instead.…”

Section: Discussionmentioning

confidence: 99%

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

Bomati

Noel

2005

Plant Cell

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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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Apo and Holo Structures of an NADP(H)-dependent Cinnamyl Alcohol Dehydrogenase from Saccharomyces cerevisiae

Cited by 51 publications

References 50 publications

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis

Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8

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

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