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Title: Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum

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Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
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Journal Article: Published Article
Journal Name:
Plant Physiology (Bethesda)
Additional Journal Information:
Journal Name: Plant Physiology (Bethesda); Related Information: CHORUS Timestamp: 2017-06-12 20:30:30; Journal ID: ISSN 0032-0889
American Society of Plant Biologists
Country of Publication:
United States

Citation Formats

Jun, Se-Young, Walker, Alexander M., Kim, Hoon, Ralph, John, Vermerris, Wilfred, Sattler, Scott E., and Kang, ChulHee. Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum. United States: N. p., 2017. Web. doi:10.1104/pp.17.00576.
Jun, Se-Young, Walker, Alexander M., Kim, Hoon, Ralph, John, Vermerris, Wilfred, Sattler, Scott E., & Kang, ChulHee. Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum. United States. doi:10.1104/pp.17.00576.
Jun, Se-Young, Walker, Alexander M., Kim, Hoon, Ralph, John, Vermerris, Wilfred, Sattler, Scott E., and Kang, ChulHee. 2017. "Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum". United States. doi:10.1104/pp.17.00576.
title = {Enzyme activity and substrate specificity of the major cinnamyl alcohol dehydrogenases in sorghum},
author = {Jun, Se-Young and Walker, Alexander M. and Kim, Hoon and Ralph, John and Vermerris, Wilfred and Sattler, Scott E. and Kang, ChulHee},
abstractNote = {},
doi = {10.1104/pp.17.00576},
journal = {Plant Physiology (Bethesda)},
number = ,
volume = ,
place = {United States},
year = 2017,
month = 6

Journal Article:
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  • Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of different types of the mammalian enzyme. Two avian forms were recently reported, and the authors now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digest. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to themore » human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehnydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. In conclusion, the amphibian enzyme allows a rough positioning of the divergence of the alcohol dehydrogenase classes, shows that the class I type is widesprread in vertebrates, and functionally conforms with greater variations at the substrate-binding than the coenzyme-binding site.« less
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  • The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of -hydroxyacids to -hydroxyacids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of , -dicarboxylates with hydrophobic -chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length -carboxylate groups. These enzymes stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins leads to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, themore » small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between 2 and 3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence, but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These structural and kinetic results will help engineer new stereospecific hydro-lyase enzymes for chemo-enzymatic syntheses.« less
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  • The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC], a plant specific deformylase, has been determined at a resolution of 2.4 {angstrom} (1 {angstrom}=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substratemore » binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr{sup 178} as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr{sup 2} in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr{sup 178} to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr{sup 178} can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.« less