Structure of Aldehyde Dehydrogenase C (AldC) mutant (C291 A) from Pseudomonas Syringae
Lee SG, Harline K, Abar O, Akadri SO, Bastian AG, Chen HS, Duan M, Focht CM, Groziak AR, Kao J, Kottapalli JS, Leong MC, Lin JJ, Liu R, Luo JE, Meyer CM, Mo AF, Pahng SH, Penna V, Raciti CD, Srinath A, Sudhakar S, Tang JD, Cox BR, Holland CK, Cascella B, Cruz W, McClerkin SA, Kunkel BN, Jez JM. The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase. J Biol Chem. 2020 Oct 2;295(40):13914-13926. [1]
Function of your protein
serve as a metabolic housekeeping enzyme due to their ability to detoxify aldehydes, which are highly reactive compounds generated through cellular. They can also scavenge aldehyde from lipid peroxidation and convert them to a less chemically reactive carboxylic acid. Aldc additionally play an important role in ethanol metabolism via oxidation of acetaldehyde into acetate, metabolism of polyamine, and plant cell wall ester biogenesis. The bacterial pathogen Pseudomonas syringae is the organism that is causing mutation in the host.
In order to determine the substrate preference of AldC, a panel of 23 molecules including short to long chain aliphatic aldehydes was used to screen for enzymatic activity. Spectrophotometric assays of AldC identified aliphatic aldehydes of 5-9 carbon length as substrates. It turns out that the 8 carbon carbon substrate is the preferred aliphatic aldehyde substrate in this case Octanal, which is surrounded by aromatic rings. On the comparison table, data suggest that short 2-4 carbon aldehydes, branched aliphatic aldehydes and larger aromatic aldehydes are poor substrates.It turns out that 8 carbon carbon substrate is the preferred aliphatic aldehyde substrate in this case Octanal, which is surrounded by aromatic rings. To evaluate the nicotinamide cofactor preference for AldC, the activity of the enzyme was tested either with octanal and NADP+ or NAD+, which showed a distinct preference for NAD+. octanal has the highest Kcat value and lowest Km value which increases its efficiancy.
Biological relevance and broader implications
The bacterial pathogen peudomanas syringae is used as a model for understanding microbial evolution, how host and pathogens interact and bacterial virulence mechanisms. P.syringae utilizes several strategies to manipulate hormone signaling in its host plants. These interactions help agriculturalists to detect diseases in plants and how to protect crops from being invaded by these harmful pathogens. In order to suppress host defenses and promote disease development, P. syringae produces a wide variety of virulence factors including auxin Indole-3-acetic acid (IAA) synthesis, whose production is implicated in pathogen virulence. PtoDC3000 synthesizes IAA using an uncharacterized pathway that requires indole-3-acetaldehyde dehydrogenase. pseudomonas species evolved to grow under unfavorable environmental conditions such as high temperature, low oxygen or water availability. They also evolve metabolic diversity and plasticity to use a variety of nutrient courses to detoxify organic chemicals that are toxic and produce multiple specialized metabolites. P.syringae develops bacterial virulence mechanisms to survive in the adverse environmental conditions of the phyllosphere. Learning about plant pathogen interactions is very important because these interactions have a profound effects not just on the plants but on humans as well. Potential development of inhibitors for P. Syringae could be useful for pathogen control in agriculture.
The article also mentions that there is a variety of pseudomonas species that have evolved to grow under unfavorable environmental conditions such as severe nutrient limitation, extreme temperatures, low oxygen or water availability and high salinity. Additionally, they have evolved metabolic diversity and plasticity to use a variety of nutrient sources like nitrogen and carbon to detoxify organic chemicals and to produce specialized metabolites.
Important amino acids
amino acids in AldC.
Asn 159, Glu 257, Gly 288, Cys 291
Cys 291 is the one that mutates to Ala 291
(binding site)
Ile 155, Asn 159, Lys 182, Gly 219, Ile 233, Ser 236, Ala 239, leu 242, Glu 257, leu 258, Gly 259, Cys 291, Glu 391, Phe 393
Nicotinamide ring is helped in place by van der Waals interactions with Leu 258, Leu 419, and Phe 456 and a hydrogen bond from the backbone carbonyl of Leu 258 to the NH2 group of the cofactors. Polar interactions between the adenine ribose ring and side chains of Lys 182 and Glu 185 contribute to NAD+ binding. Interaction of Glu 185 with the 2' hydroxyl group of the adenine ribose determine the cofactor specificity as AldC is not able to accomodate the 2 phosphate of NADP(H) sterically.
Trp 160 Tyr 163, Trp 450, Phe 456, Tyr 458, met 114, leu 118
Apolar interactions dominate the octanal binding in the hydrophobic substrate binding pocket. A cluster of aromatic residues and two nonpolar residues (Methionine and Leucine) peovides hydophobic environment that accommodates octanal and other aliphatic aldehydes. The substrate binding site forms an aromatic box for adaptable apolar ligand interaction.
To examine the contribution of the active site residues, a series of site directed mutants targeting residues in the NAD(H) binding site and the octanal binding site were generated. All the 31 mutants were expressed in E.coli and purified using nickel-affinity and size exclusion chromatographies. The enzyme activity screening showed that mutation of the catalytic residues in the NAD(H) binding site and octanal binding site resulted in enzyme with less than 1% of WT specific activity.
The article mentions that the unambigous electron densities for NAD+ and octanal define how lingands bind to the AldC C291A mutant and is the one that indicates the location of the substrate and cofactor-binding sites.
Structural highlights
has two domains, hydrophobic and hydrophilic regions.
The N-terminus of Aldc contains a central beta sheet surrounded by alpha helices which forms the NAD(H)-binding site.
Additionally, around the C-terminus there is a mixture of alpha and beta domains which includes the cysteine residue and forms the aldehyde binding site. A small three stranded beta sheet domain facilitates aligomerization. There is an interdomain linker region that connects the N and C terminal domains of Aldc.
The Amino acid sequence highlight how the catalytic cysteine and residues of the NAD(H)-binding site are highly conserved with major variations in the substrate binding site which leads to functional differences in Aldehyde dehydrogenases. Amino acids in AldC in the oligomerization domain change the electrostatic surface charge and the surface topology.
shows the hydrophilic(green) and hydrophobic (purple) tails...
Other important features
of the NAD(H) binding domain provides extensive polar and apolar interactions that position the nicotinamide ring of NAD+ in proximity to the C291A point mutation.Its main function is to bind NAD+ cofactor and contribute to substrate binding. The cleft found in the protein is where the active site is, the binding cleft contains the octanal binding amino acids. This fold is also called a beta alpha beta fold because the beta strands participate in the formation of a beta sheet.
Glu 391 Mutation, loss of Glu 257 side chain removed the internal interaction that helps position the amine group of the nicotinamide and changes at this residue could change the orientation of the nicotinamide group for hydride transfer.
Cysteine is the most important catalytic residues
between amino acids. This helps to understand how the polar amino acids
References
- ↑ Lee SG, Harline K, Abar O, Akadri SO, Bastian AG, Chen HS, Duan M, Focht CM, Groziak AR, Kao J, Kottapalli JS, Leong MC, Lin JJ, Liu R, Luo JE, Meyer CM, Mo AF, Pahng SH, Penna V, Raciti CD, Srinath A, Sudhakar S, Tang JD, Cox BR, Holland CK, Cascella B, Cruz W, McClerklin SA, Kunkel BN, Jez JM. The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase. J Biol Chem. 2020 Aug 12. pii: RA120.014747. doi: 10.1074/jbc.RA120.014747. PMID:32796031 doi:http://dx.doi.org/10.1074/jbc.RA120.014747
