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Structure
The AAT D22T mutant consists of two chains that are made up of alpha-helices. Unlike normal aspartate aminotransferase, it does not contain any beta-sheets. the D222T variant has 7438 non-hydrogen atoms and 6608 macromolecules with no bound ligands. It has a length of 414 amino acids. It also has a wavelength of 0.97856 Å. When looking at the homodimer structure, the two distinctive chains can be seen (chain A and chain B). In , multiple conformations can be seen; however, the crystal packing restrains its small domain and its movement (NLM). is not restrained like chain A, making its small domain more mobile. The hydrogen-bonding networks differ between the chains, as well as the different mutants.
The aspartate aminotransferase enzyme is a complex dimeric protein with a high molecular weight (Ovchinnikov). Structurally, aspartate aminotransferase is a homodimer, meaning it is formed by non-covalently bonded identical proteins, usually two. It contains alpha-helices, as well as beta-sheets. Functionally, aspartate aminotransferase is critical for glutamate regulation, the biosynthesis of such turns L-aspartate or L-cysteine to L-glutamate (UniProt). Glutamate is the major excitatory neurotransmitter in the central nervous system in vertebrates. The reactions catalyzed can be seen as: (L-aspartate + 2-oxoglutarate = oxaloacetate + L-glutamate) or (L-cysteine + 2-oxoglutarate = 2-oxo-3-sulfanyl propanoate + L-glutamate).
Function
Amino acids are the foundation of proteins and are also the building blocks of life. Understanding the residues of these amino acids and their roles is one of the most challenging aspects of modern biology. The synthesis and breakdown of these building blocks is called amino acid metabolism. The enzyme aspartate aminotransferase cytoplasmic D222T mutation or AAT D222T mutant plays a very interesting part in that process. Putting the name into layman’s terms, AAT is the enzyme, while D222T is the actual single mutation of that enzyme. This mutant is in close relation to H143, T139, and H189 mutants. It is classified as a transferase, which means they catalyze the transfer of specific functional groups from one molecule to another.
In order to get the proteins essential in life, all creatures use the same 20 amino acids.
This pathway accounts for 10 to 15 percent of the total energy production in organisms.
Of the 20 amino acids, 9 are considered essential, meaning they are received through dietary sources.
The remaining 11 amino acids are obtained through the amino acid metabolism process. Since
aspartate aminotransferase is considered an aminotransferase, it, for example, will take the nitrogen
-containing amino groups (+H3N-) from amino acids to ketoacids like pyruvic acid or alpha-ketoglutarate,
which is an example of a transamination reaction. This type of reaction is reversible, meaning
if you take that same enzyme and run it the other way, you will get the molecule you started with.
Amino acid metabolism is important to other pathways in a multitude of ways. For example,
taking glutamate into consideration again, it is a very unique amino acid. It is the only amino
acid that doesn’t have to transfer its nitrogen contain amine group to another molecule, and it
does that through oxidative deamination. Oxidative deamination typically occurs in the
mitochondria where the enzyme glutamate dehydrogenase takes the amino group and adds an
oxygen group from H2O to form alpha-ketoglutarate as stated before. The hydrogens from
glutamate get transferred to NAD+ and will eventually produce NADH. The ammonia left in the
mitochondria will then be able to convert into urea, through the process of the urea cycle. That
is just an example of how amino acid metabolism connects pathways.
For amino acid metabolism to perform the way that it does, there has to be the presence
of PLP. Pyridoxal 5′-phosphate (PLP) is an incredibly important multifunctional enzyme
cofactor used to catalyze many chemical reactions involved in the amino acid metabolism process.
D222T mutations are a type of mutation that causes a decrease in the electrophilicity, as well as the kcat
(substrate turnover), while increasing the affinity. This has a large effect on enzyme activity.
How this works is through enzymes that are dependent on pyridoxal 5’-phosphate optimizing specific chemical reactions by modulating electronic states of PLP in distinct active site regions (Proteopedia). An extended hydrogen-bond network from aspartate aminotransferase coupled to pyridoxal 5’- phosphate through its pyridinyl nitrogen will influence the electrophilicity of the cofactor.
PLP-dependent enzyme aldimines have multiple ionizable sites, the most significant of which, in terms of the D222T mutant, is the PLP-N1 position or pyridine nitrogen, where PLP is deprotonated. It has a pKa value of ~5.8 PLP’s different protonation states is a key part of its role in catalysis. The D222T mutant’s x-ray crystal structure does have an extended hydrogen bond network to PLP-N1. That network promotes the protonation of PLP. D222T is not directly hydrogen bond to PLP-N1 but there is a Thr-222 coupled with structural water that connects the two. When PLP is protonated, the electronic sink effect of it changes significantly. Research done by individuals at the Universities of Toledo and Tennessee have found that when the proton protonating PLP-N1 is moved to in D222T, it decreases the kcat by ~99%, as well as Km. That is a very significant drop in value and it decreases the catalytic efficiency of the enzyme, meaning it decreases the turnover amount of substrate molecules that transform into their intended products per unit time. To go along with this, the L-Asp affinity does increase, which refers to the greater interaction or binding strength of molecules.
Relevance
This enzyme is commonly found in E. coli that is in sus scrofa or swine, meaning wild boar, hog, or pig.
Getting into the taxonomy of sus scrofa is important. Classified as even-toed ungulates, they fall under the Animalia kingdom, Mammalia class, and the Chordata phylum. Originating from North Africa and Eurasia, the wild swine has the widest-ranging mammal in the world. They inhabit a tremendous array of habitats all over the planet; so much so that they are considered an invasive species. On record, there are about 599 specimens and 4 subspecies (boldsystems). These subspecies include Sus scrofa cristatus, Sus scrofa domesticus, Sus scrofa scrofa, and Sus scrofa taivanus. These creatures have a long history of association with humans, which has led to them becoming the most widespread suiform. Due to this expansion, it has been seen that there is a massive change in gene expression and gene regulation during the adaptation to these new habitats (biomedcentral).
Their divergence around 1.5 million years ago, as well as human-mediated dispersal, has led to the current results in their gene flow. It also resulted in very different minor allele frequencies at millions of genomic locations (biomedcentral). It is not normally recognized that due to domestication and selection by humans, the phenotypic variation of sus scrofa is incredibly large. Due to this, as time went on, this species has seen genes rapidly evolve and involve themselves in host defense, immunity, and sensory perception. Due to the great changes in gene regulation within Sus scrofa, this likely resulted in mutations such as the D222T mutant. The gene in question that affects the amino acid metabolism and produces aspartate aminotransferase within the sus scrofa population is called Glutamate Oxaloacetate Transaminase 1 (GOT1). GOT1 is conserved and present in many other species including Homo sapiens. In sus scrofa, the gene is typically expressed in the heart and a few other tissues. Not only does it play a role in amino acid metabolism, but it is also present for the urea and tricarboxylic cycles, as well as a slew of other processes. The GOT1 gene regulates general cell metabolism through the usage of carbohydrates and amino acids to meet nutrient requirements in the body. Part of that regulation, as stated before, includes amino acid metabolism through informational coding of aspartate aminotransferase.
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