Dihydroxy_acid_Dehydratase_1

Holo-Structure of Dihydroxy-acid Dehydratase (DHAD) Complex with [2Fe-2S] cluster (PDB ID: 5ZE4) from Arabidopsis Thaliana

Created by: Samantha Reiss

The holo-structure of the dihydroxy-acid dehydratase (AthDHAD) complex with a [2Fe-2S] cluster (PDB ID: 5ZE4) from Arabidopsis thaliana is a biomolecule bound to several ligands (Mg2+, Fe2/S2 Cluster, sulfate, and acetate) (1). The protein is catalytically active with a Fe2S2 cluster bound but not with a Fe4S4 which acts as a competitive inhibitor to prevent substrate and product binding (2). It has potential as a natural-product herbicide for crop development, working against weed immunity (3). This protein is not wild type and has K559A/K560A double mutations. . It is a chloroplastic dihydroxy-acid dehydratase that synthesizes L-isoleucine from 2-oxobutanoate through methyl cleavage and amine transfer (4).

AthDHAD also catalyzes the third step of the branched chain amino acid (BCAA) biosynthesis by dehydrating and tautomerizing 2,3-dihydroxy-isovalerate and 2,3-dihydroxy-3-methylvalerate to the corresponding 2-keto acids. This pathway is a frequent target for current herbicide development since it is not present in mammals. To create better crop yield and evade herbicide resistance, new herbicides need to have novel activity. Since farms and gardens are competitive environments for plants and microorganisms, microbes need to produce chemicals that kill plants to increase their growing space (5). By genome mining for these natural microbial herbicide products and determining their specific biological activities, a fungal product was discovered as an effective bioactive natural herbicide. It works on distinct components of the BCAA pathway and may be more effective than currently available herbicides since pathway disruption causes plant death. The estimated agricultural demand in ten years is sixty percent higher than today due to population growth. However, the annual agricultural growth is expected to decline in the next thirty years. Many countries are running out of suitable farm land, making increased efficiency for future crop production important for feeding the world (6).

For the purification and investigation of DHAD, Escherichia coli (E. Coli) was used as the expression vector. AthDHAD is an asymmetric monomer that can form a dimer when activated. It was crystallized in monomeric form through sitting-drop vapor diffusion in an anaerobic box created by equilibrating the proteins against a reservoir and by forming a cross-section electrostatic map. The entire protein is crystallized besides the chloro- segment at the N-terminus of the protein and a transit peptide. There are no prosthetic groups or substrate/product attachments in the crystallized structure, and no ligands were present in the crystal structure that were used merely to induce crystallization and serve no biological function in this biomolecule (1, 3).

Val-496 and Ile-177 in AthDHAD are important for protein function They form a larger hydrophobic pocket than succinyl glutamate-semialdehyde dehydrogenase (AstD) (PDB ID: 4KNA), the enzyme mutated with the fungal herbicide resistance gene. This allows aspterric acid, an inhibitor of BCAA, into AthDHAD’s the active site but not AstD’s whose smaller opening leads to aspterric acid immunity (4,5). The exact interaction between aspterric acid and AthDHAD is not yet determined. New herbicide production is important due to increasing weed resistance. By discovering both a new herbicide and its gene product target in plants, a new area of crop modification allows for the creation of plants resistant to weed-killing herbicides so that only the weeds are targeted with treatment (4).

The ligands bound are functionally important for substrate binding to the molecule. These ligands bind in distinct places within the protein. The protein’s associated metal ions are Fe2S2 and Mg2+. Mg2+makes the interior of the active site chamber positively charged, and the Fe2S2 inorganic cluster increases the catalytic properties of the active site. Mg2+ ionically binds Asp-140 and Glu-463 through ionic interactions, and Fe2S2 binds to Cys-66, Cys-139, and Cys-211 through disulfide bonds. The protein is only catalytically active with a Fe2S2 bound as Fe4S4 acts as a competitive inhibitor to prevent substrate and product binding (2). Sulfate binds Arg-25, Ser-31, Lys-30, and Asp-46 through ionic interactions and hydrogen bonds. Sulfate occupies the catalytic site, and acetate stabilizes structure. Important active site residues for substrate interaction are Ile-177, Phe-181, Leu-465, Ser-489, and Val-178 (3).

The Expasy database was used to determine that DHAD has a molecular weight of 61148.09 Da and an isoelectric point of 5.44 (7). The protein is an asymmetric monomeric biomolecule which was crystallized at a resolution of 2.11 Å (1). The primary structure of DHAD contains 573 residues with an arrangement of both hydrophobic and hydrophilic amino acids (1). The secondary structure of this protein is composed of 10 α-helices, 25 segments of random coils, and 22 β-strands (8). The α-helix-dominated structure is important because of the mixed polarities of the sequence; the α-helix allows the hydrophobic residues to be pushed to the inside of the globular structure of the protein, while still allowing the polar residues to face outwards. The tertiary structure of DHAD, due to the nonpolar residues from the helical structures facing inwards contains a hydrophobic core and an active site where several ligands bind to influence substrate binding. Ionic interactions that are important for the enzyme structure include Asn-140 and Gln-463. Its identified domain is a N-terminal transit peptide with a peptide sequence that directs the protein to the correct organelle and allows for transport across membranes; the motifs have yet to be classified (4). As stated previously, the quaternary structure of DHAD is a monomer, lacking interactions with other subunits.  

The Basic Local Alignment Search Tool (BLAST) database compares protein sequences and calculates the statistical significance of matches to a protein structure based on E-value (10). This finds relationships between protein sequences and identifies members of protein families. The E value is determined by the presence of gaps, or lack of homology, between two primary structures. A lower E value represents more closely related primary structures, i.e. fewer gaps. E values below 0.05 are considered significant. The Dali Server compares the 3D structure of a protein to others in the protein data bank to reveal similarities in 3? structure. It provides a quantitative Z score to signify the degree of structural similarity. A Z-score over 2 is considered significant.

D-xylonate dehydratase in holo-form (PDB ID: 5OYN) derived from Caulobacter vibrioides (strain ATCC 19089/CB15) has similar primary and tertiary structures as DHAD (1). It is in the 6-phosphogluconate dehydratase family; both D-xylonate dehydratase and DHAD are part of the Ilv/ED dehydratase protein family whose members are involved in various biosynthetic and carbohydrate metabolic pathways (9). The E value of DHAD and D-xylonate dehydratase in their respective complexes was 2e-66, indicating very similar sequences (11). The comparison of DHAD and D-xylonate dehydratase yielded a Z-score of 43.1, which indicates the tertiary structures are very similar.

The largest similarity between D-xylonate dehydratase and DHAD in their respective ligand complexes is the dominant helical structure. D-xylonate dehydratase’s associated ligands are Mg2+, Fe2S2, and a KCX residue modification for L-peptide linking; the metal ligands of D-xylonate dehydratase and DHAD are shared.  Although very similar, there are structural differences that exist between the two biomolecules.D-xylonate dehydratase's quaternary structure is a homotetramer, composed of four homologous subunits with dihedral global symmetry, whereas DHAD is best known as a monomer. Due to substrate similarity and there being 4 substrates, the total structural weight of D-xylonate is 262696.59 Da with 2400 residues instead of 573 (1). The general lyase activity is in both DHAD and D-xylonate dehydratase. Their prefered carbohydrate substrates differ slightly in structure with D-xylonate being a longer chain that contains more hydroxyl and less methyl groups (10). The active site of D-xylonate dehydratase is located on the interface of two subunits, utilizing residues from the N-terminal domain of one subunit and the C-terminal domain of the dimeric counterpart while DHAD’s active site does not involve the monomers terminal sequences (9). DHAD’s active site is more exposed to the polar environment and residues compared to multimeric similar proteins such as D-xylonate dehydratase. This makes the aspterric acid interaction even more favorable with DHAD than with other similar proteins evolved plants may feature from developed resistance.

Aspterric acid inhibits DHAD activity but not AstD (12). These similar structures are both influential in plant biology. The more closed active site of AstD prevents aspterric acid inhibition. Many weeds have this gene activated, making them immune to common herbicide treatment that use aspterric acid. By mutating plants to also feature AstD instead of DHAD, more intense herbicides can be used to kill the weeds without killing the plants being farmed.To create these mutations, data science and genomics are used both to search fungi genomes for genes that provides fungal poison immunity and to determine variations between the plant and fungal genes at these loci. This approach is known as resistance gene-directed genome mining.

Resistance gene-directed genome mining allows for a better understanding of critical enzymatic and molecular functions which can have diverse applications not only in agriculture but also in human health. New antibiotics and cancer-fighting drugs can be formed by identifying metabolic regulation mechanisms, finding novel gene functions, and using similar tools as with AthDHAD manipulation. Finding a resistance gene protecting a microorganism from an antibacterial product can help identify the genes involved in producing that anti-bacterial compound, leading to new antibacterial medicines (5).