Assessment of a putative proton relay in Arabidopsis cinnamyl alcohol dehydrogenase catalysis.

Extended proton relay systems have been proposed for various alcohol dehydrogenases, including the Arabidopsis thaliana cinnamyl alcohol dehydrogenases (AtCADs). Following a previous structural biology investigation of AtCAD5, the potential roles of three amino acid residues in a putative proton relay system, namely Thr49, His52 and Asp57, in AtCAD5, were investigated herein. Using site-directed mutagenesis, kinetic and isothermal titration calorimetry (ITC) analyses, it was established that the Thr49 residue was essential for overall catalytic conversion, whereas His52 and Asp57 residues were not. Mutation of the Thr49 residue to Ala resulted in near abolition of catalysis, with thermodynamic data indicating a negative enthalpic change (ΔH), as well as a significant decrease in binding affinity with NADPH, in contrast to wild type AtCAD5. Mutation of His52 and Asp57 residues by Ala did not significantly change either catalytic efficiency or thermodynamic parameters. Therefore, only the Thr49 residue is demonstrably essential for catalytic function. ITC analyses also suggested that for AtCAD5 catalysis, NADPH was bound first followed by p-coumaryl aldehyde.

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof.

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.