Introduction
The Aldo-Keto Reductase (AKR) superfamily is a diverse group of enzymes that catalyze redox transformations. These transformations play crucial roles in various biological processes, including biosynthesis, intermediary metabolism, and detoxification. AKRs are found in a wide range of organisms, from microorganisms to humans, and are characterized by a conserved structural motif and the ability to reduce a broad spectrum of substrates.
Structural Features of AKR Enzymes
The (β/α)8 Barrel Motif
AKR proteins share a common (β/α)8 barrel, also known as a TIM-barrel motif (triosphosphate isomerase). This structural motif represents a compact yet adaptable scaffolding. It allows for structural variations required for binding a chemically-diverse range of carbonyl substrates. The active site of AKRs is located at the C-terminal face of the barrel. It is optimized for high-affinity interaction with pyridine nucleotides in the absence of a canonical Rossman fold.
The (β/α)8 motif has wide functional utility. It can be utilized to bind redox active cofactors, and metals, to oligomerize into quanternary arrangements that can form active site interfaces or it could be used as a gated barrel for channeling reaction intermediates.
A distinguishing feature of the (β/α)8 barrel fold is the presence of the active site at the C-terminus. This feature is considered to be indicative of a common ancestry. The ready interconversion of the substrate specificity of (β/α)8 proteins in a single round to random mutations affecting the C-terminus further supports their common ancestry from progenitor proteins of broader substrate specificity.
Structural Diversity and Family Identity
While AKRs share the (β/α)8 barrel structure, they also exhibit structural diversity that contributes to their functional specificity. This diversity arises from variations in loops and helixes that interrupt the α/β barrel fold. These variations bring structural identity to individual families. The “hot spot” for such variations is the region between the 7th and the 8th β-strands of the barrel.
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For example, aldehyde and aldose reductases (AKRs 1A and 1B) have a long loop between β9 and α7 that opens and closes above the bound nucleotide. This facilitates tight binding to NADPH. The length of this loop is variable among members of the family. In some AKRs, e.g. hydroxysteroid dehydrogenases (AKR1Cs), its small size results in the absence of opening or closing movements.
Conserved Features
Aldo-keto reductases are ancient proteins that share a common conversed (β/α)8 barrel structure and a conserved pyridine nucleotide binding site.
Another conserved feature of (β/α)8 proteins is the presence of a phosphate binding site. Approximately two-thirds of the established (β/α)8 barrel enzymes utilize substrates or cofactors that contain phosphate group. In the canonical (β/α)8 structure, the central inner ring of 8 parallel β-strands in a hyperboloid structure is wrapped by an outer envelop consisting of 8 external α-helices. This generates a highly symmetrical arrangement of secondary structural elements. The structure of the inner β-strand barrel is constrained, whereas, the arrangement of α-helices is more variable.
Functional Aspects of AKR Enzymes
Catalytic Activity and Substrate Specificity
Using pyridine nucleotide as cofactors, most AKRs catalyze simple oxidation-reduction reactions. A characteristic feature of AKRs is their ability to catalyze aldehyde or ketone reduction. Because these proteins lack metal or flavin cofactors, they are relatively inefficient as alcohol dehydrogenases.
Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. The proteins encoded by Akr genes catalyze a variety of metabolic oxidation-reduction reactions ranging from the reduction of glucose, glucocorticoids and small carbonyl metabolites to glutathione conjugates and phospholipid aldehydes.
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Role in Metabolism and Detoxification
In this capacity, the AKRs function as independent metabolic units or as inter-linked components of metabolic pathways. In these pathways, these proteins work in collaboration with other carbonyl-metabolizing enzymes such as aldehyde and alcohol dehydrogenases, cytochrome P450s (CYPs) and glutathione S-transferases (GSTs).
The conversion of aldehydes to alcohols, which results in the reduction of the polar carbonyl group, and decreases the overall chemical (but not necessarily the biological) reactivity of the molecule, therefore, represent one mode of inactivation and detoxification. Several drugs, pharmaceuticals, foods, and pollutants are reactive carbonyls and aldehydes or are converted to carbonyls during in vivo metabolism (e.g. by CYP450 catalyzed conversions).
Cofactor Preference
Most AKRs prefer NADPH over NADH. In metabolically active cells, NADP+ is mostly in the reduced form. Therefore, reduction is favored over oxidation. The NADPH/NADP+ ratio is reflective of the synthetic capacity of the cell and is kinetically and thermodynamically dissociated from the NAD+/NADH ratio, which is mostly regulated by rates of glycolysis and respiration. Hence AKRs can accomplish their tasks of metabolism and detoxification without being affected by fluctuations in the cofactor ratio due to changes in metabolic rate and capacity.
Tight binding to NADPH provides some AKRs (e.g. aldose reductase) a thermodynamic advantage for achieving the transition state without placing much energetic demand on the substrate. Because most of the energy required for carbonyl reduction is derived from nucleotide, not carbonyl, binding, even substrates that are loosely bound to active site residues are reduced with high efficiency. As a results, aldose reductase reduces a wide range of aldehydes.
AKR1C Isoforms: Structure, Function, and Role in Disease
The AKR1C1-AKR1C4 genes are located on chromosome 10 p15-p14 and comprise of 12 exons. And the average molecular weight of enzymes is estimated to be 34-42 kDa. These enzymes share a high percentage of amino-acid sequence identity that ranges from 84 to 98%. The AKR1C isoforms play pivotal roles in NADPH dependent reductions. Therefore, the enzymes are highly related to malignant cancer involve NADPH reductive progress like PCa, breast cancer, and etc.
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AKR1C4 is mainly liver-specific and recently it has been proved to be related to manic/hypomanic irritability in males. The AKR1C3 protein is also known as PGF synthase that catalyzes the conversion of prostaglandins H2 and D2 into PGF2α and 9α,11β-PGF2α respectively. It has the highest catalytic efficiency of the AKR1C enzymes to interconvert testosterone with Δ4-androstene-3,17-dione. The enzyme will also reversibly reduce 5α-DHT, estrogen and progesterone to produce 3α-androstanediol, 17β-estradiol and 20α-hydroxprogesterone, respectively.
While AKR1C4 and AKR1C3 are almost exclusively in the liver and prostate respectively, AKR1C1 and AKR1C2 are most prominent in the mammary glands includes breast cancer, endometrial cancer, colorectal cancer. AKR1C2, is also known as bile-acid binding protein and DD2, has lower catalytic efficiencies but preferentially reduces 3-ketosteroids. AKR1C2 preferentially reduces DHT to the weak metabolite 5α-androstane-3α,17β-diol (3α-diol) without conversion of 3α-diol to DHT in the PC-3 cell line.
AKR1C3 and Cancer
There are also significant correlations between the expression levels of AKR1C3 and CRPC. And AKR1C3 overexpression is proved to be a promising biomarker for PCa progression. Positive AKR1C3 immunoreactivity was also extensively present in both adenocarcinoma and squamous cell carcinoma arising from the lung and the gastroesophageal junction.
Elevated levels of AKR1C3 expression in CRPC over PCa have been reported. High affinity binding of DHT to the AR initiates androgen-dependent gene activation and contributes to PCa development and progression. In the prostate, 5α-DHT can be reduced to 3α-diol through the action of reductive 3α-HSDs. Between the two major 3α-HSD isozymes, AKR1C2 and AKR1C3, in human prostate, both isozymes catalyze the reversible reduction of 5α-DHT activity toward the weakly androgenic metabolite 3α-diol, which is recognized as a weak androgen with low affinity toward the AR. AKR1C1, which is associated with the HSD3B pathway of DHT metabolism, expressed at higher levels than AKR1C2, catalyzes the irreversible conversion of DHT to 3β-diol.
AKR1C3 is known to be abundantly expressed in breast cancer tissues, and high levels are often associated with adverse clinical outcome. AKR1C3 is capable to produce intratumorally testosterone and 17β-estradiol by reducing the androgen precursors and estrogen, respectively. The local conversion of less potent hormones to more potent ones will lead to nuclear receptor activation and tumor progression. Therefore, AKR1C3 has recently been identified as a potential therapeutic target in both CRPC and ER-positive breast cancer. AKR1C3 is responsible for the reduction of PGD2 to11β-PGF2α, both of which were reported to demonstrate similar affinities toward their cognate receptor, Prostaglandin receptor (FP receptor). And the action of FP receptor ligands results in carcinoma cell survival in breast cancer.
The expression of AKR1C1 and AKR1C2 was found reduced in tumorous breast tissue. Then in vitro studies had shown that progesterone metabolites can regulate PR-negative breast cell tumor formation and growth as well as tumor regression and maintenance of normalcy. Progesterone is degraded to its metabolite 20α-DHP by AKR1C1 and to 3α-HP by AKR1C2. These metabolites promote suppression of cell proliferation and adhesion. These 20α-DHP and 3α-HP bind to specific plasma membrane receptors, separate from classical HRs, and influence anti-proliferative functions on mitosis, apoptosis, and cytoskeletal and adhesion molecules.
AKRs and Drug Resistance
An emerging theme is the role of AKRs in cancer chemotherapeutic drug resistance. Among the mechanisms of resistance, metabolic inactivation by carbonyl reduction is a major cause of chemotherapy failure that applies to drugs bearing a carbonyl moiety. AKR1C3 does also catalyze the inactivation of the anticancer drug doxorubicin. Doxorubicin undergoes metabolic detoxification by carbonyl reduction to the corresponding C13 alcohol metabolite, doxorubicinol. In comparison to doxorubicin, doxorubicinol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes.
Induction of AKR1C1 and AKR1C3 has been shown to efficiently abolish the efficacy of daunorubicin chemotherapy for leukemic U937 cells by metabolizing both DNR and cytotoxic aldehydes derived from ROS-linked lipid peroxidation. Aldo-keto reductase 1C3 (AKR1C3) is also linked to doxorubicin resistance in human breast cancer which resulted from activation of anti-apoptosis PTEN/Akt pathway via PTEN loss. The biochemical basis for resistance to cisplatin in a human ovarian cancer cell line has also been reported to be due to overexpression of the AKR1C1 though the underlying mechanism has not been revealed yet.
Non-Catalytic Functions of AKR1C3
Previous studies about AKR1C isoforms mostly revealed their biological function in an catalytic-dependent role. However, their non-catalytic functions have remained elusive until Yepuru M. Notably, it was recently reported that AKR1C3 can regulate AR activity in a catalytically independent role. Yepuru M. and his co-workers found that as an enzyme converts androstenedione to testosterone, AKR1C3 also acts as a selective coactivator for the AR to promote CRPC growth. AR can interact with AKR1C3 and get recruited to the ARE on the promoter of androgen responsive genes. Thus, recruits related cofactors leading to activation of transcription on reduction of target genes. And while the full-length of proteins is necessary to mediate AKR1C3’s enzymatic functions, amino acids 171-237 were sufficient to mediate the AR activation.
Another example of a catalytically independent role of AKR1C3 on AR activity was found in regulating Siah2 stability. Ubiquitin ligase Siah2 was reported to enhance AR transcriptional activity and PCa cell growth. Interactions between steroid biosynthetic enzymes and steroid receptors may be exceedingly complex and involved in a variety of hormone-dependent cancers.
AKR1C3 and Radiation Sensitivity
Firstly, there was evidence that after short-term and long-term cadmium exposure, the expression of AKR1C1 was elevated which implies the role of ARKs in cell sensitivity. Then studies found that AKR1C3 siRNA significantly enhanced cell radio sensitivity. Consistently with this, overexpression of AKR1C3 enhances resistance of cancer cells to radiation.
AKR1C2 and Metastasis
AKR1C2 is mostly involved in the process of metastasis. Li et al. (2016) identified two powerful genes in the liver cancer metastasis process, AEG-1 and AKR1C2. And then AEG-1 was proved to promote metastasis through downstream AKR1C2 and NF1 in liver cancer.
Inhibitors of AKR1C
Several types of AKR1C1 inhibitors have been identified, including, benzodiazepines, steroid carboxylates, phytoestrogens, derivatives of pyrimidine, phthalimide, anthranilic acid and cyclopentane, flavones and ruthenium complexes. Notably, 3-bromo-5-phenylsalicylic acid, an inhibitor designed based on the structure of AKR1C1 in ternary complex with NADP+ and DCL, its phenyl group targets a non-conserved hydrophobic pocket in the active site of the enzyme lined by residues Leu54, Leu308 and Phe311, resulting in a 21-fold improved potency (Ki = 4 nM) over the structurally similar AKR1C2. Structure between AKR1C1 and AKR1C2 is rather similar, only differs by one active-site residue (Leu54 versus Val54). Therefore, the selectivity of inhibitors targeting AKR1C1 and AKR1C2 is rather low, and newly designed inhibitors that mostly interact with Leu54 in AKR1C1 are needed as to improve the selectivity over AKR1C2. Derivatives of BPSA, 3-chloro-5-phenylsalicylic acid (Ki = 0.86 nM), is 24-fold more selective for AKR1C1 over AKR1C2.
AKR1C3 is inhibited by several classes of AKR1C3 inhibitors, including cinnamic acid, non-steroidal anti-inflammatory drugs (NSAIDs) and their derivatives, steroid hormone analoges, flavonoids, cyclopentanes, benzoic acids, progestins, baccharin analogs, ruthenium complexes, and the most widely used anti-diabetes drugs, sulfonylureas. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. Critical concern in exploiting AKR1C3 inhibitors is the cross inhibition of AKR1C subfamily members, as they have high amino acid sequence identity and structural similarity.
Evolutionary Aspects
Aldo-keto reductases are ancient proteins that share a common conversed (β/α)8 barrel structure and a conserved pyridine nucleotide binding site. To-date more than 100 members of this family have been described. These proteins are found in all phyla ranging from prokaryotes, protozoans, and yeasts to plants, animals, and humans. They are believed to have originated from a now extinct multifunctional ancestor by divergent evolution involving gene duplication and subsequent evolutionary variances in substrate binding and preferences.
Based on the level of sequence homology, the AKR superfamily is divided into 15 families and some families are further divided into subfamilies. Members of each family share more than 40% homology with each other and less than 40% with members of any other family. Mammalian AKRs fall within 3 well-defined families (AKR1, 6 and 7). These proteins are widely distributed in tissues and most cells express several AKRs. In humans, 13 different AKR proteins have been identified that fall within the 3 major families of mammalian AKR.