| D-bifunctional protein deficiency | |
|---|---|
| Other names | 17β-hydroxysteroid dehydrogenase IV deficiency |
| Specialty | Medical genetics |
D-Bifunctional protein deficiency is an autosomal recessive peroxisomal fatty acid oxidation disorder. Peroxisomal disorders are usually caused by a combination of peroxisomal assembly defects or by deficiencies of specific peroxisomal enzymes. The peroxisome is an organelle in the cell similar to the lysosome that functions to detoxify the cell. Peroxisomes contain many different enzymes, such as catalase, and their main function is to neutralize free radicals and detoxify drugs. For this reason peroxisomes are ubiquitous in the liver and kidney. D-BP deficiency is the most severe peroxisomal disorder,[1] often resembling Zellweger syndrome.[2]
Characteristics of the disorder include neonatal hypotonia and seizures, occurring mostly within the first month of life, as well as visual and hearing impairment.[3] Other symptoms include severe craniofacial disfiguration, psychomotor delay, and neuronal migration defects. Most onsets of the disorder begin in the gestational weeks of development and most affected individuals die within the first two years of life.
YouTube Encyclopedic
-
1/2Views:9 9395 632
-
Aspartyl Proteases: Mechanism & Inhibition
-
Introduction to Chemical Biology 128. Lecture 07. DNA, RNA, and Cancer.
Transcription
About 20 years ago scientists discovered that there was an aspartyl protease that was an essential enzyme to the replication of the HIV virus, and so immediately upon this discovery this protein became the target of enzyme inhibition. If you were assigned to the task to come up with an inhibitor of this enzyme the first thing you'd want to know is what's the mechanism of the normal mode of action of aspartyl proteases. And so let's take a look at that here. Aspartyl proteases had long been known. They're actually enzymes that are found in the digestive system. They contain two aspartyl groups in the enzyme-active site. That's why they're called aspartyl proteases. One of them is going to be protonated and serves as a general acid. The other will be unprotonated and can serve as a general base. Ah, because of their acidic groups, these aspartyl groups are quite acidic, they are able to function at low pH, and so that's why they're, ah, capable of operating in the stomach where the pH can be kite- quite low, and a prototypical example is the enzyme, pepsin. The HIV protease is- consists of two chains. Each of them are about 99 kDa in size and one chain will deliver one aspartyl group and the other chain will deliver the other aspartyl group into the active site that we're going to see in just a moment. Substrate specificity, it was known that the, ah, HIV protease was particular at cleaving the peptide bond between an aromatic amino a- acid residue and proline. So here's the scissile bond that undergoes, ah, fragmentation, undergoes cleavage in the, ah, enzyme-active site. Here's the aspartyl protease, ah, ah, that active site with the aspartyl protease on position 25 from one chain and 25' that comes from the other chain. The, one of these aspartate groups is protonated and the other one is unprotonated. So we've got the general base on the right, the general acid on the left. Water is going to add into this peptide bond and so we're going to use the general base to activate that nucleophilic addition and then we're going to use the general acid to protonate the oxygen of the carbonyl group. That creates what the, is a tetrahedral intermediate. That tetrahedral intermediate is actually going to have two hydroxyl groups. It's, ah, ah, basically a dihydrate at that, ah, tetrahedral carbon. And we've reversed the role now of these two aspartate groups. The one that was the acid has become the base. The one that was the base has become the acid. And so we're just going to turn around the electron flow starting with the aspartate group that's the base, deprotonate and then kick out, eliminate in this β elimination step the proline carbon- nitrogen bond. So that's the electron flow for that turned around step. This is actually the rate-determining step and so we're going to want to know what's the- in trying to think about an inhibitor, we're going to wanna pay a lot of attention to the transition state structure for this rate-determining step. That immediately yields the product. The product is, um, going to be this, ah, amino group, the carboxylic acid group of what used to be the peptide bond. The one aspartyl group has now become acidic again and the other one has become basic, and so we have sort of the classical example of bifunctional catalysis. Let's take a look at the three-dimensional structure of the HIV protease. It's a fascinating structure. We'll see the two different protein chains are colored, ah, differently in this space-filling model and if we take a view or we spin this around there's a side-on view, look at the front view, we can see that there's a cavity that is where the active site is and this cavity goes all the way through from one side to the other. [clears throat] It's known that the way that this active site binds its substrate is by having these two flaps basically open up so they can, ah, sort of, ah, spring open and, ah, collect the substrate, engulf it in the enzyme-active site and subject it to catalysis. Let's take a look at how the substrate binds and where those aspartyl groups are. Here's the, ah, here's a space-filling model where you can see now the substrate in yellow and now a wire-frame model that you can see that and as we zoom in on this we'll be able to see where those, ah, aspartyl groups are. So let's look down here and we can see that there are the two aspartyl groups. They're basically dangling right into the carbonyl group of where the substrate is going to occupy. So we can put the substrate in place and... there it is. The substrate is, ah, shown in yellow. We see those two aspartyl groups, ah, hanging into the carbonyl group of that peptide bond that undergoes fragmentation. You get an idea here I think of how this protein serves to both create that cavity that can bind to the substrate to be able to recognize those two residues that it recognizes but also to position basically rigidly hold those aspartyl groups in place. Here's another view. We can spin this around. There's a side view. There's a side view. You can keep going and you can get back around and you can see those two groups hanging right in that carbonyl of the, ah, of that yellow substrate molecule. That substrate's actually an inhibitor. The inhibitor that was discovered is going to be shown here on this slide, but let's look at that transition state for that rate-determining step because really that's what we're trying to mimic. We're going to come up with a reversible inhibitor of this aspartyl protease and in trying to think about what the enzyme is doing, what it's binding most tightly is the, ah, transition state leave- in the rate-determining step and so we can take a look at what that transition state looks like by thinking about the electron-flow arrows for how that, ah, dihydrate breaks down and so we can see that there's going to be a diminishing negative charge there developing negative charge over here. The key thing is is that we can see that there should be some tetrahedral bond with hox- hydroxyl groups on them and the proline should be, ah, nearby. And so as scientists thought about coming up with an inhibitor, they really wanted to create something that mimicked this geometry but had a lot greater stability so that it couldn't break down, and that's exactly what they produced when they came up with the molecule that had the name JG-365. The structure of JG-365 is shown over here and a couple of key things that JG-365 has going for it is first of all rather than having a nitrogen in this position, which would be a good leaving group and could be protonated by the general acid, it has a CH2 group and so this bond becomes, this carbon-carbon bond, becomes completely stable and is unable to undergo the normal bond-breaking step through the general base catalyzed β elimination process. That's one thing it has going for it and also it's got the basic mimicking of the tetrahedral geometry, that t- that tetrahedral geometry that's found in the transition state structure. And so the JG-365 is effective because it's stable to hydrolysis and it has the geometry of that transition state structure. So if you do a super position, we can imagine how this molecule is going to, ah, basically be superimposable directly on the transition state structure that we have, ah, drawn for you here. The only difference is in this CH2 group that connects to the proline, which is provided, that CH2 group is provided because it gives that stable bond. So that was a solution to the problem. It binds quite tightly, 0.66 m- nM is the dissociation constant and so this turned out to be quite an effective inhibitor of the HIV virus.
Classification
DBP deficiency can be divided into three types:[4]
- type I, characterized by a deficiency in both the hydratase and dehydrogenase units of D-BP
- type II, in which only the hydratase unit is non-functional
- type III, with only a deficiency in the dehydrogenase unit
Type I deficient patients showed a large structural modification to the D-BP as a whole. Most of these individuals showed either a deletion or an insertion resulting in a frameshift mutation. Type II and III patients showed small scale changes in the overall structure of D-BP[6]. Amino acid changes in the catalytic domains or those in contact with substrate or cofactors were the main cause of these variations of D-BP deficiency. Other amino acid changes were seen to alter the dimerization of the protein, leading to improper folding. Many mutations have been found in the gene coding for D-BP (HSD17B4) on the q arm two of chromosome five (5q23.1) in Homo sapiens, most notably individuals homozygous for a missense mutation (616S).[4]
D-BP Protein
The D-bifunctional protein is composed of three enzymatic domains: the N-terminal short chain alcohol dehydrogenase reductase (SDR), central hydratase domain, and the C-terminal sterol carrier protein 2 (SDR).[1]
The DBP protein (79kDa) also known as "multifunctional protein 2", "multifunctional enzyme 2", or "D-peroxisomal bifunctional"enzyme", catalyzes the second and third steps of peroxisomal β-oxidation of fatty acids and their derivatives .[citation needed]
A non-functional D-BP protein results in the abnormal accumulation of long chain fatty acids and bile acid intermediates. The D-BP protein contains a peroxisomal targeting signal 1 (PTS1) unit at the C-terminus allowing for its transport into peroxisomes by the PTS1 receptor. Inside the peroxisomes, the D-BP protein is partially cleaved exclusively between the SDR and hydratase"domains.[1]
DBP is a stereospecific enzyme; hydratase domain forms only (R)-hydroxy-acyl-CoA intermediates from trans-2-enoyl-CoAs.[4] D-BP is expressed throughout the entire human body, with the highest mRNA levels in the liver and brain. The hydrogenase and hydratase units of DBP exist as dimers, necessary for correct folding and therefore function of the enzyme.
Genetic
The D-BP gene (HSD17B4), found on the long arm of chromosome 5, consists of 24 exons and 23 introns and is over 100kb in size. Exons 1-12 code for the SDR domain, 12-21 for the hydratase domain, and 21-24 for the SCP2 domain. Transcription is regulated at 400 basepairs upstream of the transcription start site.[1]
The missense mutation G16S is the most common mutation that leads to D-BP deficiency. In a 2006 study in which 110 patients were tested, 28 had this frameshift mutation. The second most frequent mutation was the missense mutation N457Y which was seen in 13 of the 110 patients. Type I patients showed only deletions, insertions, and nonsense mutations were identified, most leading to shortened polypeptides. Most type II patients show missense mutations in D-BP hydratase unit as well as some in-frame deletions. Type III"individuals commonly show missense mutations in the coding region of the dehydrogenase domain.[4]
Chemistry
Enzymatic activity of D-BP fails if the protein cannot effectively bind the cofactor NAD+, as shown in the G16S mutation. Glycine 16 forms a short loop and creates a hole for the adenine ring of NAD+ to enter. Other amino acid side chains alter the shape of this loop due to steric hindrance, and prevent proper NAD+ binding. Other mutations that exist are due to incorrect polypeptide folding. L405 (leucine located at residue 405) located in the substrate binding domain of the hydratase 2 unit, plays an important role in binding CoA ester moiety. One mutation seen in D-BP deficiency patients is caused by a leucine to proline substitution. This breaks the hydrophobic interactions necessary for proper substrate binding with CoA esters.[4]
Diagnosis
The most common clinical observations of patients with D-bifunctional protein deficiency include hypotonia, facial and skull dysmorphism, neonatal seizures, and neuronal demyelination.[5] High levels of branched fatty acids, such as pristinic acid, bile acid intermediates, and other D-BP substrates are seen to exist. Reduced pristinic acid β-oxidation is a common indicator of D-BP deficiency.[1] D-BP can be distinguished from Zellweger Syndrome by normal plasmalogen synthesis. Recent studies in D-BP knockout mice show compensatory upregulation of other peroxisomal enzymes in absence of D-BP such as palmitoyl-CoA oxidase, peroxisomal thiolase, and branched chain acyl-CoA oxidase.[1]
References
- ^ a b c d e f Möller G, van Grunsven EG, Wanders RJ, Adamski J (January 2001). "Molecular basis of D-bifunctional protein deficiency". Mol. Cell. Endocrinol. 171 (1–2): 61–70. doi:10.1016/s0303-7207(00)00388-9. PMID 11165012. S2CID 29712091.
- ^ Itoh M, Suzuki Y, Akaboshi S, Zhang Z, Miyabara S, Takashima S (March 2000). "Developmental and pathological expression of peroxisomal enzymes: their relationship of D-bifunctional protein deficiency and Zellweger syndrome". Brain Res. 858 (1): 40–7. doi:10.1016/S0006-8993(99)02423-3. PMID 10700594. S2CID 11224543.
- ^ Buoni S, Zannolli R, Waterham H, Wanders R, Fois A (January 2007). "D-bifunctional protein deficiency associated with drug resistant infantile spasms". Brain Dev. 29 (1): 51–4. doi:10.1016/j.braindev.2006.06.004. PMID 16919904. S2CID 617635.
- ^ a b c d e Ferdinandusse S, Ylianttila MS, Gloerich J, Koski MK, Oostheim W, Waterham HR, Hiltunen JK, Wanders RJ, Glumoff T (January 2006). "Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis". Am. J. Hum. Genet. 78 (1): 112–24. doi:10.1086/498880. PMC 1380208. PMID 16385454.
- ^ van Grunsven EG, Mooijer PA, Aubourg P, Wanders RJ (August 1999). "Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency". Hum. Mol. Genet. 8 (8): 1509–16. doi:10.1093/hmg/8.8.1509. PMID 10400999.
External links
This page was last edited on 29 April 2024, at 22:58