Sepiapterin Reductase (4HWK) from Homo sapiens
Created by: Cammie Wheeler
Sepiapterin reductase (SPR) (PDB ID: 4HWK) from homo sapiens is an oxidoreductase enzyme that reduces the carbonyl groups in both pterin and non-pterin substrates. It has a molecular weight of 124,946.09 Da and an isoelectric point of 8.70 (1). It is closely related to short-chain dehydrogenase/reductase (SDR) family of enzymes (2).
SPR plays an important role in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for reactions involving phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase (3). It catalyzes the final step in two possible biosynthetic pathways for BH4. Firstly, it reduces the two carbonyl compounds found in 6-pyruvoyl-tetrahydropterin to hydroxyl groups, resulting in the formation of BH4 (4). Alternatively, it operates in a salvage pathway where it reduces sepiapterin to dihydrobiopterin (BH2), which can then be further reduced to BH4 (3). Both of these reactions are NADP dependent and will not occur in the absence of the coenzyme (3). A deficiency of SPR activity results in a shortage of BH4, since it cannot be synthesized without the enzyme. Low BH4 levels can lead to hyperphenylalaninemia as well as dopamine and serotonin deficiencies, all of which have been linked to negative side effects to the central nervous system such as nausea, headache, drowsiness, insomnia, tremors, and even psychosis (4). This is because BH4 is necessary for the synthesis of neurotransmitters, catecholamines, and indolamines that play vital roles in the nervous system (3). Therefore, because SPR is necessary for the successful and efficient synthesis of BH4, it is an important enzyme for proper neurological function.
The quaternary structure of SPR is a homodimer (5), so it contains two identical monomers with the same function. This subunit structure is shared by the SPR found in mus musculus(PDB ID: INAS). The formation of the dimer from two identical monomers results in two viable substrate-binding sites, allowing for multiple reductions to occur simultaneously (5).
The formation of the dimer results in a bundle of four α-helices, two from each monomer (5). This folded structure is stabilized by a combination of hydrogen bonds between the α-helices and ionic interactions between the Asn-141and Asn-116 residues from each respective monomer (5). Two binding pockets are formed from loops stabilized by the H-bond interactions between nearby α-helices and β-sheets (5). These pockets are 15 Å deep, making them especially conducive to interactions with pterin and carbonyl groups (5). The SPR proteins found in both mice and humans share a similar tertiary structure. This fact is supported by the results of a Dali Server search, which compares the intramolecular distances between proteins using a “sum of pairs” method. It uses this information to compare the proteins’ tertiary structures, and generates a Z score to represent the relative similarity of their three-dimensional shapes. A Z score higher than two indicates a significant degree of similarity between the folding patterns of two proteins. The Dali Server produced a Z score of 42.0 for the comparison of the human and mouse SPR proteins, which shows that they share similar folding patterns (6). Additionally, many members of the SDR family appear to have a similar three-dimensional composition, suggesting that this particular folding pattern is highly conducive to the reduction reactions these enzymes catalyze (2). The two binding sites are where the substrates attach to SPR in order for reduction to occur and are, therefore, the sites of all of its enzymatic activity. The weak forces mediating the tertiary structure are essential to the enzyme’s functionality, as they result in the formation of the active sites necessary for SPR to successfully catalyze reactions.
The secondary structure of human SPR contains 41% α-helices and 15% β-sheets and has a total of 11 α-helices and 11 β-sheets (7). Comparatively, the mouse SPR contains 47% α-helices and 17% β-sheets, but has the same 11 α-helices and 11 β-sheets (7). Both proteins have an α/β- structure and the center of each monomer consists of seven parallel β-sheets sandwiched between two groups of 3 α-helices (5). Interestingly, while there are no antiparallel β-sheets in SPR, the parallel β-sheets in each monomer are arranged antiparallel to those found in the other monomer (5). Overall, the secondary structure contributes significantly to the three-dimensional shape as the nature and interaction of the helices and sheets determines the folding patterns seen in the tertiary structure.
The amino acid sequence of SPR enzymes is highly conserved between species and both proteins contain 261 residues within their sequence (5). The primary structure of mouse and human SPR has 74% homology (8). Furthermore, the results of a PSI-Blast analysis revealed an E score of 2 e -135, indicating a significant degree of sequence similarity (9). The PSI-Blast finds proteins with similar primary structures to a given protein of interest and assigns E scores to each protein with sufficient similarity. The E scores are based on the degree of sequence homology and the number of gaps, which are portions of sequence present in the protein of interest, but not in the comparison protein. An E value less than .05 is considered significant. Therefore, the results of the Blast analysis support that the two species’ respective proteins share a similar primary structure. The high degree of conservation across species suggests that the arrangement of and presence of particular residues in the sequence plays an important role in this type of enzyme’s proper function.
Research involving the crystallized structure of SPR has discovered several residues that play significant roles in the function of the enzyme. Its reduction capacity is dependent on the presence of the NAPD coenzyme and several functionally important residues. During a reduction reaction, NADP binds to SPR at residues 14-42 at a specific orientation, which is facilitated by H-bonds formed between the NADP and Lys-175, Asp-70, and Leu-71 (5). Additionally, the side chains of Arg-43, Leu-71, and Leu-127 sandwich the adenine moiety of NADP in place near the binding pocket (5). Once stably in place, the NADP is able to interact with a substrate bound to this active binding site (5). The hydrophobic residues Leu-105, Leu-159, Tyr-165, Trp-168, Tyr-171, Met-106, and Cys-160 make up the cavity that forms this substrate-binding pocket (5). Because of their position at the C-terminal end of the parallel β-sheets in each monomer, Asp-158 in mice and Ser-158 in humans are able to interact with pterin substrates bound in that region (5, 2). They play the important role of stabilizing pterin substrates at the proper location and orientation within this binding site for successful reduction to occur by forming H-bonds with the pteridine ring (5, 2). Once both the coenzyme and substrate are in place, the nicotimamide ring of NADP transfers a hydride ion to the substrate’s carbonyl group, resulting in the carbonyl group adopting a negative charge (5). Tyr-171, the central active site residue, transfers a proton from the hydroxyl group located in its side chain to the now nucleophillic carbonyl carbon, thereby reducing the substrate (5). Tyr-171 is located within the binding pocket, and its orientation places it at an optimal distance from the carbonyl group on the substrate for this transfer to occur (5). Lys-175 and Ser-158 facilitate this reaction by stabilizing the resulting tyrosinate structure after it loses its proton (2). Overall, theSer-158-Tyr-171-Lys-175 triad is highly conserved in SPR and a similar sequence is found in SDR proteins (3), which demonstrates that the cooperative function of these residues plays an important role in the enzyme’s reduction activity. The functional importance of these components of the primary structure has also been demonstrated by research involving the inhibition of these residues. For example, the binding of an analogue to Asp-158 prevents SPR from reducing larger pterin derivative substrates because the pteridine ring is not stabilized properly within the binding site (5). However, the reduction of smaller, non-pterin substrates is not inhibited by blocking this residue as these substrates do require further stabilized by H-bonds to interact successfully with SPR (5). Additionally, the binding of inhibitors to Tyr-171 or the NADP ligand prevents all reduction activity (5), as they both play vital roles in reduction reaction.
Similarly, research into anti-inflammatory sulfa drugs shows that they can inhibit SPR activity, and, subsequently, reduce physiological BH4 levels (4). To investigate this effect, researchers solved the crystal structure of SPR while it reacted with NADP and sulfapyridine, a common type of sulfa drug. They determined that the three substances interact to form a ternary complex in which the sulfa drug blocks SPR’s active binding site (4). Specifically, the sulfonamide and nitrogen groups within the heteroaromatic ring found in these drugs form H-bonds with the Ser-158, Tyr-171, and Asp-258 (4). In doing so, the drug prevents these functionally important residues from interacting with and reducing substrates. Additionally, the pyridine ring found in these drugs stacks on top of the nicotinamide ring in NADP (4), blocking its ability to participate in hydride transfer to a nearby substrate. Because these sulfa drugs have inhibitory effects on SPR, they reduce the levels of BH4 available to produce important signaling molecules used in nervous system function. Therefore, their use can cause the type of detrimental effects on motor control and cognition that commonly occur as a result of BH4 shortages.