Sandbox Reserved 1565

Function(s) and Biological Relevance

(IMPDH) is the enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway, converting inosine monophosphate (IMP) to xanthosine monophosphate (XMP) with the reduction of nicotinamide adenine dinucleotide (NAD). Organisms that undergo the purine nucleotide biosynthetic pathway have IMPDH, including humans. This particular form of IMPDH described and highlighted comes from the recently studied fungus Ashbya gossypii ^[1]. Additional ligands of IMPDH include nicotinamide adenine dinucleotide (NAD), acetate (ACT), and guanosine-5’ monophosphate (5GP), and guanosine-5'-diphosphate (GDP). IMPDH is a regulator of the intracellular guanine nucleotide pool amount and helps control control cell division and proliferation, and therefore related to malignant transformation, tumor cell proliferation, and intracellular and extracellular pathogenic infections. Purine dinucleoside polyphosphates are found to bind to the Bateman domain of Ashbya gossypii IMPDH to allosterically regulate the catalytic activity by competing against purine mononucleotides^[2].

Broader Implications

The disease pathophysiology is extensive as the improper IMPDH regulation may lead to uncontrolled cell division and proliferation, affecting the immune system's ability to fight off pathogens and signal tumor cells to apoptose. Therapeutic studies using dinucleoside polyphosphates may allosterically regulate the inhibition of IMPDH activity. Dinucleoside polyphosphates have physiological functions including cell division, neurotransmission, apoptosis, vasoconstriction, platelet aggregation, and cellular process variety enhancement from DNA replication to repair^[3]. Selected IMPDH inhibitors composed of dinucleoside polyphosphates may be used to make IMPDHs targets for immunosuppressive, antiviral, and anticancer drugs^[4], with antibacterial and chemotherapeutic strategies feasibile^[5].

Structural Highlights and Structure-Function Relationships

The shows alpha helices and beta sheets. The Ashbya gossypii IMPDH is 31% helical and 15% beta sheet, with the other percentages including random coils and residue structures. The active site is located towards the C-terminus within the TIM barrel, containing 8 alpha-helices and 8 beta sheets. The CBS (Bateman) domain is recognized in the secondary structure through its beta-alpha-beta-beta-alpha pattern.

IMPDH quaternary structures include multiunit complexes, such as , extended octamers, and compacted octamers. These quaternary structures are created through the binding of multiple subunits of structures (monomers), that are strengthened and structurally formed through hydrogen-bonding, cysteine-cysteine disulfide bonds, and hydrophobic interactions. Different quaternary forms of IMPDH relate to the kinetic favorability of the IMPDH mechanism as Bateman domain allosteric binding sites and competitive nature changes with unit composition. The secondary structure of IMPDH's Bateman domain is folded, so its antiparallel beta-sheets are surrounded by the alpha helices (one on each side) to form a globular tertiary structure.

This helps show the Van der Waals interactions and areas for movement within the structure. The ability for monovalent cations to move within the charged tunnel with the phosphate chain (TIM barrel) directly relates to activation levels. The phosphate chain relates to the ligands that further interact with the binding site to form the covalent intermediate, E-XMP*.

The is within the interior of the molecule as the hydrophilic residues are able to interact in a physiological environment. The hydrophobic region of Gly361 and Gly383 interact with the main chain phosphate (TIM), further allowing monovalent cation movement. Hydrophilic regions contain amino acid residues that hydrogen-bond, some conserving tertiary structure and others relating to necessary interactions in the active site (see below).

Acetate ions, G5P, and GDP molecules are of IMPDH. NAD serves a function as a ligand as is necessary in the hydrolysis of IMP. The phosphates of the G5P, GDP, and NAD (not pictured) ligands interact through hydrogen bonds and hydrophobic interactions (depending on the amino acid residue; see active binding site below). The monocovalent cations that pass through interact with the phosphates as they activate IMPDH.

The IMPDH includes Arg (325), Asn (306), and Asp (272). This is represented by the solid purple structures in the image. This triad is important as it makes cysteine more reactive as a nucleophilic component. This conserved cysteine, Cys334, (Cys331 in human type II IMPDH) induces binding after becoming more reactive (Cysteine shown in image with "active binding site" link).

The includes the Bateman domains, which are components within the TIM barrel. Binding occurs after the catalytic triad makes cysteine more reactive. The cysteines that become more reactive are shown in green in the image, and are closely related to the active binding site. Asp259 (blue) hydrogen bonds with the ribose hydroxyls of NAD (nicotinamide region), and Ser315 (blue) hydrogen bonds to the ribose phosphate through hydroxyl groups. Gly361 and Gly383 (orange) have hydrophobic interactions with the phosphate of the ligand NAD. Other important interactions include Tyr403 hydrogen bonding to ribose phosphate (NAD), and Glu402 and Glu440 hydrogen bonding with the IMP purine ring.

IMPDH is not positively or negatively strong as an entire protein, as shown by this view. There are positive and negative components within the structure, but a relatively neutral substance is better received in this mechanism due to a physiological environment. Negatively-charged glutamic acid and positively-charged histidine within this enzyme play a role within the covalent bindings in the mechanism. Covalent binding is necessary to form the covalent intermediate after NAD is reduced (after interacting with the active site residues).

This enzyme's contains mostly protein (brown) with no solvent. The dark pink RNA regions coincide with G5P and GDP ligands as they contain ribose groups. NAD, containing two ribose groups, (not pictured) is another ligand that is necessary in the hydrolysis of IMP in the mechanism. The green acetate ions are anions that function as ligands as as intermediate-step metabolites in the mechanism. Monocovalent cations travel through and activate IMPDH as anionic acetate ions buffer the system.

Energy Transformation

IMPDH is activated by monovalent cations, such as K+ and Na+, within the triose-phosphate isomerase (TIM) barrel. The Arg325-Asn306-Asp272 catalytic triad (Arg322-Asn303-Asp274 in human type II IMPDH) works inter-dependently and synergistically in the TIM barrel active site to make the nucleophilic component, cysteine, highly reactive to form a temporary covalent bond with the substrate^[6]. Substrates bind randomly to IMPDH as the hydride transfer is quick and NAD is reduced to hydrolyze the covalent intermediate within the enzyme-substrate complex. A covalent intermediate, E-XMP*, is formed, which decreases the energy needed in later nucleophilic and covalent catalysis steps^[7]. Based on normal physiological conditions, the IMPDH mechanism is often not kinetically favorable. The Bateman domains within the TIM barrel are composed of cystathionine beta-synthase motifs that perceive metal ion concentration, cellular energy status, and ionic strength; and will allosterically regulate IMPDH activity^[8]. Eukaryotic IMPDHs have three nucleotide-binding sites in the Bateman domain that allosterically modulate catalytic activity. These three nucleotide-binding sites bind adenine/guanine dinucleoside polyphosphates, and the affinity for these sites increases for these dinucleoside polyphosphates as the activity of IMPDH increases. Purine dinucleoside polyphosphates compete with purine mononucleotides within these sites, so the Bateman domain sites make IMPDH more sensitive to inhibition^[9]. Enzyme catalysis is able to finish with the energy need reduced as the covalent bond is broken later in the reaction to regenerate the enzyme.