Bioinformatics Analysis of Aspergillus Flavus Urate Oxidase - Research Paper Example

Aspergillus flavus Urate Oxidase: Molecular Bioinformatics Analysis Introduction The DNA sequence: 5’cctggcgcgccagcagctgatcgagactgt-3’ was assessed using BLASTn, a search engine of National Center for Biotechnology Information (NCBI 2010). This search program identified a gene from the microbe Aspergillus flavus, that encodes the enzyme urate oxidase , as the closest matching DNA sequence, with (60%) sequence homology to the full-length gene. This gene and its encoded protein were assessed further using bioinformatics databases to explore important structural and functional parameters of this gene and its encoded protein. BLASTp was used to identify the amino acid sequence of the urate oxidase gene. Comparative genomics assessment was also carried out using NCBI databases to explore the conservation of this gene and its evolutionary relationships among diverse species. Urate oxidase (UAO), also called uricase, is an enzyme of Aspergillus flavus that functions in the metabolism of purines. The reaction catalyzed by this enzyme is the conversion of uric acid to allantoin. Although conserved in evolution, apes and humans lack this enzyme and secrete uric acid directly in the urine without further processing to allantoin (Li et al. 2006). The protein gene product of Aspergillus flavus uricase is a tetramer of four identical subunits with a molecular weight of 32,000 daltons (NCBI 2010). The DNA sequence of the uricase gene of Aspergillus flavus was determined by cloning and sequencing a complementary DNA (cDNA) prepared from messenger RNA (mRNA) transcripts of the uricase gene in this microorganism (Legoux et al. 1992). The following essay is the product of data on the genetic origins of this DNA sequence with respect to gene identification by sequence homology, predicted amino acid sequence, protein structure and functional assessment and comparative genomics results obtained by the use of bioinformatics to assess nucleotide sequences. DNA sequence analysis The DNA sequence of the uricase gene of Aspergillus flavus was originally determined by cloning and sequencing a complementary DNA (cDNA) prepared from messenger RNA (mRNA) transcripts of the uricase gene in this microorganism. The uricase gene was found to encode an open reading frame (ORF) comprised of 302 codons (Legoux et al. 1992). The gene contains two introns; the cDNA and the gene are identical except for the absence of the intron sequences in the cDNA. The details of gene structure were accessed via BLASTn database records (NCBI 2010). The gene contains a typical eukaryotic structure (NCBI 2010). A TATA box that comprises the primary binding site for RNA polymerase II is located approximately 25 bases upstream from the transcription start site. The first exon contains the initiator AUG translation start site. There are three exons separated by two stretches of non-coding intervening sequences called introns. The exon/intron boundaries share the canonical GT/AG splice signals as wellas an internally recognized splice recognition sequence. A polyadenylation signal is located in the terminal exon. Following transcription, the primary transcript is capped, polyadenylated and spliced to generate a functional messenger RNA (mRNA) for translation (Prosite 2010). A comparative genomic analysis of the uricase gene from Aspergillus flavus and that of other species by means of sequence alignment studies to identify overall DNA sequence homology of the uricase genes encoded by different species indicated that the uricase gene of A. flavus is distantly related to the gene identified in mammalian species (Legoux et al. 1992). It is distantly related also to the uricase genes of Drosophila and soybean. Specifically the uricase gene from A. flavus shares 42-43% homology to the mammalian uricase genes and 41-42% homology to the soybean and Drosophila uricase genes (Legoux et al. 1992). Protein analysis The UOA gene of A. flavus encodes a predicted protein sequence of 302 amino acids, based on the presence of 302 codons in the exons of this gene (NCBI 2010). The codons identified in the coding regions can be used to generate a predicted amino acid sequence without direct protein sequencing, which is a difficult and time-consuming methodology. The predicted protein sequence can be compared to proteins encoding similar sequences of amino acids using the BLASTp database search engine (NCBI 2010). This search indicated that the UOA protein of A. flavus contains considerable sequence homology to uricase genes from other species. Comparative analyses have also shown that highly conserved regions of protein sequence are found in uricase genes of widely divergent species (Li et al. 2006). Specifically, there are two regions within the protein that are very highly conserved in all uricase enzymes. One of these regions of highly conserved amino acid sequence identifies a consensus sequence pattern for uricase enzymes, termed motif 1 (Li et al. 2006). This sequence motif contains the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser and is present in all uricase enzymes analyzed thus far. Some variation within this consensus sequence has been observed. In A. flavus, the sequence reads Val-Leu-Lys-Ser-Thr-Asn-Ser; however, all amino acid substitutions are conservative with respect to R group components. A second consensus sequence pattern in uricase enzymes, termed motif 2, has also been identified (Li et al. 2006). This sequence reads Ser-Pro-Ser-Val- Gln-Lys/His/Asn-Thr-Leu-Tyr. Once again the modified form of this consensus sequence in uricase of A. flavus reads: Ser-Ala-Ser-Val-Gln-Ala-Thr-Met-Tyr. In this motif, the sequence changes in A. flavus are not highly conserved changes (Li et al. 2006). Within the cell, uricase enzymes are located in the peroxisomes. This protein localization site is consistent with the presence of a peroxisome transfer signal in the carboxy terminal region of uricase from A. flavus. These sequences comprise the carboxy terminus of the enzyme and read: Ser-Lys-Leu. This triplet amino acid sequence has been found to function as a peroxisome transfer signal sequence (Legoux et al. 1992). The cloned cDNA corresponding to the UOA gene from A. flavus has been expressed in E. coli to produce high level expression of the uricase protein (Legoux et al. 1992). Moreover, the recombinant enzyme was shown to have biological properties indistinguishable from that of the naturally produced enzyme of A. flavus, a condition necessary for therapeutic application. Post-translational processing of the uricase enzyme of A. flavus is comparable to that of other species in that the amino terminal methionine is removed and the adjacent amino acid is acetylated by N-acetyltransferases (Li et al. 2006). Data obtained from mass spectrophotometry of uricase protein from A. flavus showed that serine is the site of N-acetylation of this protein (Prosite 2010). This process does not occur in the synthesis of the recombinant protein in E. coli since the bacteria lacks enzymes with this substrate specificity; however, the absence of acetylation appears to have no effect on the enzymatic function of this protein and may be required only to stabilize the protein and block degradation within the cell (Li et al. 2006). Structure/function analysis of urate oxidase from A. flavus Urate oxidase is an enzyme that plays an important role in purine metabolism in many eukaryotic species (Legoux et al. 1992). Purines are nitrogenous bases found in nucleic acids most commonly in the form of adenine and guanine. The enzyme urate oxidase (UOX) is a component of the purine degradation pathway that metabolizes uric acid, a purine degradation product, to 5-hydroxyisourate (HIU), an intermediate in purine metabolism that then forms the more stable metabolic product allantoin, which is associated with the release of carbon dioxide (Legoux et al. 1992). The catalytic step requires molecular oxygen and involves a hydroxylation reaction. The biochemical steps of this enzyme catalyzed pathway were determined by chemical studies of enzyme-substrate reaction components and products. These studies were enhanced by nuclear magnetic resonance (NMR) studies of enzyme substrate complexes (Prosite 2010). In addition, critical information on the three dimensional structure of the native protein and enzyme substrate complexes and intermediates have been obtained by the use of protein crystallographic studies. X-ray crystallographic studies have been carried out on this enzyme to elucidate the structure/function relationships of this protein (Prosite 2010). Protein crystallization followed by X-ray diffraction represents an extremely powerful tool for the assessment of three-dimensional protein structure. The native structure of UOX was determined in this manner and compared to the structure of the protein following modifications designed to elucidate parameters of active site binding that correlate with reaction mechanism. This enzyme is a tetrameric protein comprised of four identical subunits of the protein translation product of the UOX gene (Prosite 2010). Each of the monomeric units of UOX contains an active catalytic site that is located at the interface between dimers. Each of the active sites is exposed on the surface of the tetramer; the inner component of the molecule contains a channel whose potential function is unknown. The enzymatic function of UOX requires molecular oxygen, but does not involve a cofactor, which is highly unusual for this type of enzyme (Prosite 2010). Competitive inhibition studies in conjunction with X-ray diffraction studies have provided valuable information on the structural and functional parameters of this protein (Li et al, 2006). Using the competitive inhibitor, 8-azaxanthine under dioxygen pressure has facilitated the determination of the location of molecular oxygen within the active site of this enzyme. The use of sodium cyanide to compete with dioxygen at the active site permitted the isolation of crystallized enzyme-substrate complexes to reveal the substrate binding site within the catalytic region of this complex (Li et al. 2006). Nuclear magnetic resonance studies (NMR) have been used to identify reaction intermediates of UOX. 8-azaxanthine (AZA) was used as a competitive inhibitor to define the substratrate binding site parameters of UOX (Li et al. 2006). Protein crystallization under conditions of increased oxygen pressure was also carried out to address the parameters of oxygen binding in reaction catalysis. These spectrophotometric studies have provided important information on the mechanism of catalysis by UOX (Li et al. 2006). The enzyme initially binds uric acid and then forms a complex with molecular oxygen. This mechanism of action appears to be similar to that of other oxidases that do not utilize cofactors and catalase. These data have shown that a metastable intermediate is the initial reaction product, 5-HIU. Transient-state kinetics and trapping experiments have further indicated that urate hydroperoxide is an early intermediate of this enzyme catalyzed reaction. Dioxygen activation may involve proton transfer from a deprotonated substrate to molecular oxygen (Li et al. 2006). Biochemical studies and X-ray diffraction experiments have facilitated a step by step proposed mechanism of action (Li et al. 2006). The first step in this reaction sequence involves the formation of the urate dianion from the monoanion. The next step requires the addition of dioxygen which results in the formation of 5-hydroperoxyisourate, followed by the generation of dehydrourate. Hehydrourate is then hydroxylated to produce 5-HIU. The naturally occurring substrate, uric acid, binds to the active site of the enzyme in the same location as identified inhibitors of enzyme activity (Li et al. 2006). Amino acid mutations generated in the active site of the enzyme identified important amino acids critical to substrate binding and enzyme activity (NCBI 2010). These include Thr at position 57 and Lys at position 10. On the basis of these studies, it has been proposed that water plays a role in the proton transfer mechanism of this reaction (Li et al. 2006). Comparative genomics Urate oxidase is an example of an ancient gene present in the most primitive species of eukaryotes such as yeast, highly conserved in evolution, yet it was ultimately lost from the repertoire of genes in the most recent stages of evolutionary development, including apes and humans (NCBI 2010). Comparative genomics assessments have been used to evaluate the significance of the evolutionary loss of primordial genes in the course of genetic evolution. The study of the loss of the UOX gene in higher species has provided researchers with an opportunity to assess the potential mechanisms involved in this type of genetic event. The identification of genes lost in the course of evolution can be carried out by syntenic mapping studies of gene structure in human-mouse-dog triads (Li et al. 2010). This type of assessment most commonly involves the use of a gene program called TransMap that can be used to map the structure of genes comparatively between different species. It is a cross-species mRNA alignment program that can be used to assess similarities and differences of protein coding genes among different species. The coding regions of expressed genes are scanned for the presence of inactivating mutations, principally stop codons that affect gene expression, splice mutations and frameshift mutations (NCBI 2010). This method has permitted the identification of 26 highly functional genes that have been lost in fairly recent evolutionary history (78 million years) (NCBI 2010). Among them is the UOX gene, absent only in higher primates such as apes and humans. This gene has suffered a nonsense mutation in the open reading from resulting in the insertion of a premature stop codon that prevents the translation of the protein from mRNA (NCBI 2010). This area of research more commonly focuses on the acquisition of novel genes or changes in gene structure and function that may have adaptive value. The study of gene loss is also critical to developing a coherent understanding of evolutionary mechanisms. The accumulation of genomic sequence data from a wide diversity of species has permitted the assessment of gains and losses of genes throughout evolutionary history as an important branch of comparative genomics. This process requires the identification of relevant selective pressures on the expressed proteins of specific genes and an identification of gene signatures that provide evidence of adaptive evolutionary processes. Once a gene product is no longer required for optimal fitness, it may begin to accumulate mutations that are random and ultimately affect the level of expression and/or activity of the associated gene product. The occurrence of these random inactivating mutations may therefore provide molecular evidence in support of this process. These events are associated with the presence of pseudogenes that are related in structure and function to primordial active genes, but contain mutations that prevent the expression of functional protein (Li et al. 2006). The absence of urate oxidase in humans may reflect powerful anti-oxidant activities of uric acid that may have a protective effect against the development of cancer (Legoux et al. 1992). Enzymes responsible for the breakdown of this substrate might be expected to e selected against due to their effects on removing this anti-oxidant protective effect of this substrate. Medical/therapeutic applications of UOX There are several important therapeutic uses of urate oxidase in clinical medicine. As a consequence of the absence of this enzyme in humans, uric acid can accumulate to high levels in the blood which may itself be destructive (Legoux et al. 1992). The build-up of uric acid in the form of its sodium salt in the blood is the cause of gout, a painful inflammatory characterized by the deposition of uric acid crystals in the joints. The most common treatment for gout is allopurinol, an inhibitor of xanthine oxidase, the enzyme that converts hypoxanthine to xanthine and uric acid. This treatment is not entirely effective, however, in patients with renal pathology. In these cases, the direct injection of uricase facilitates the breakdown of uric acid and its excretion to alleviate the symptoms of gout (Legoux et al. 1992). In addition, cancer patients treated with high dose chemotherapy designed to destroy DNA may accumulate purine metabolic products that can be toxic at high concentration. Treatment with UOX promotes the conversion of purine products to uric acid to alleviate this treatment side effect of chemotherapy. Hyperuricemia associated with gout and cancer chemotherapy is often treated by the therapeutic administration of the enzyme urate oxidase (Legoux et al. 1992). The traditional source of this therapeutic enzyme is Aspergillus flavus, a microbe from which the enzyme was purified by batch culture preparations for many years before the recombinant protein was created using the tools of biotechnology. The current treatment involves the use of a commercially prepared recombinant enzyme obtained from several species of yeast, including Aspergillus and Saccharomyces (Legoux et al. 1992). Conclusion Bioinformatics is an extremely important tool of molecular biology that facilitates the rapid and extensive assessment of gene structure and function. The vast scope of this technological tool encompasses the identification of single base mutations that may have profound effects on coding functions and also the comparative assessment of thousands of bases of DNA to facilitate studies of comparative genomics among distantly related species. The applications of this technology have been explored in this essay to assess a short DNA sequence with respect to its relation to the structure of known genes, the predicted amino acid sequence of the encoded protein, a comparative genomics assessment of this protein in widely diverse species and the complementary use of structural data to discern structure/function relationships of urate oxidase. The practical application of this type of assessment was also illustrated in the pharmacologic use of the cloned protein in the treatment of human disorders associated with the accumulation of uric acid. References National Center for Biotechnology Information (NCBI) Read More