The enzymes relevant to the first three steps in the predicted pathway are aldolase/transaldolase, dehydroquinate synthase, and dehydroquinate dehydratase, enzymes specified by ORFs OE1472F, OE1475F and OE1477R, respectively.
4.4.1 Aldolases EC 4.1.2.13
Aldolase is an enzyme which is capable of cleavi reaction is reversible and part of
Two distinct classes of aldolases are found in nature, which differ in their molecular properties and mechanisms (Rutter, 1964a). Class I aldolases, as represented by rabbit muscle aldolase, catalyses the cleavage of FBP through a covalently bound intermediate. The intermediate is stabilized by a Schiff base formed
between the substrate and an active site residue of the enzyme, Lys237. Enzymes which are part of this class are inactivated by borohydride (NaBH4) (Rutter, 1964b). Class II aldolases, represented by yeast aldolase, depend on divalent metal ions to stabilize the carbanion intermediate, and are inhibited by EDTA (Lebherz and Rutter, 1969).
Early studies of extreme halophiles described the distribution of class I and II aldolases in various haloarchaea. Their activity was measured in cell-free extracts in the presence of EDTA or NaBH4 (Altekar W., 1987). These studies showed that the
H. salinarum, H. volcanii and H. mediterranei enzymes exhibit the properties of class II aldolase (i.e. their activities were metal dependent), while the aldolase of H. vallismortis was classified as a class I enzyme, due to inactivation by NaBH4.
Although class I and II aldolase activities have been demonstrated in archaea (Altekar W., 1987; Dhar and Altekar, 1986; Fuchs et al., 1983; Krishnan and Altekar, 1991; Schafer and Schonheit, 1992), no genes encoding classical class I and II enzymes have been identified in any of the sequenced archaeal genomes, suggesting that archaea possess a novel type of aldolase. In 1998, Thomson et al., (Thomson et al., 1998) described a new type of FBP aldolase in E. coli, which belongs to the class I family. Although it uses a Schiff-base mechanism, its shows only low sequence similarity to other members of this class. This aldolase was originally misannotated in the E. coli genome as dehydrin (DhnA). Homologs were identified in archaea, as seen in the multiple alignments done by Siebers et al., (Siebers et al., 2001). Remarkably, M. jannaschii, A. fulgidus, H. salinarum NRC-1 and H. salinarum each possess two paralogous genes of this family, while Pyrococcus furiosus and Aeropyrum pernix each possess one gene. The enzymes from Pyrococcus furiosus and Aeropyrum pernix (Siebers et al., 2001) were expressed in E. coli, and showed preferred substrate specificity for FBP in the catabolic direction and exhibited metal-independent class I aldolase activity via a Schiff-base mechanism. Consequently, class I aldolases were grouped into two different sequence families, one with only eukaryotic members and one with both bacterial and archaeal members. The latter were designated class IA aldolase (DhnA family), possessing a catalytic lysine residue (Lys237) (Siebers et al., 2001; Thomson et al., 1998).
4.4.2 Dehydroquinate synthase EC 4.6.1.3
This enzyme catalyses the conversion of 3-deoxy-D-arabino-heptulosonate 7- phosphate to 3-dehydroquinate (DHQ) in the shikimate pathway of bacteria and eukaryotes (see figure #2, second step). The overall reaction is complex and involves alcohol oxidation, phosphate elimination, carbonyl reduction, ring opening and intermolecular aldol condensation, in a single active site (Srinivasan et al., 1963; Widlanski et al., 1989). In prokaryotes, the enzyme is encoded by a single function gene, aroB (Bentley, 1990). In some eukaryotes such as Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae, dehydroquinate synthase is produced as part of the pentafunctional AROM protein which catalyse steps 2-6 (figure #2) (Banerji et al., 1993; Charles et al., 1986).
ORF OE1475F in H. salinarum was annotated as a conserved hypothetical protein but displayed a 41% sequence similarity to MJ1294 from M. jannaschii. The latter enzyme has shown to be responsible for the synthesis of DHQ in the de novo pathway of AroAAs in M. jannaschii (White, 2004).
4.4.3 dehydroquinate dehydratase EC 4.2.1.10
A 3-dehydroquinate dehydratase is a dehydroquinate (DHQ) to form 3-dehydroshikimate (DHS) while releasing a water molecule. This enzymatic reaction is the 3rd step in the shikimate pathway (figure #2,
3rd step).
Two distinct types of 3-dehydroquinate dehydratase are found in nature which catalyze the same overall reaction but are unrelated at the sequence level and utilize completely different mechanisms (Gourley et al., 1999). Both enzyme types function as part of the shikimate pathway but while type I is found in archaea, plants and some bacteria, type II is found in bacteria and fungi. 3-dehydroquinate dehydratase from the bacterium Salmonella typhimurium was the first type I enzyme-product complex to be crystallized and its structure solved to 2.1Å resolution (Gourley et al., 1999; Lee et al., 2002). In 2004, the structure of another type I enzyme from Staphylococcus aureus was solved (Nichols et al., 2004), showing that type I enzymes belong to the (βα)8-barrel fold superfamily.
ORF OE1477R (aroD) in H. salinarum was annotated as a 3-dehydroquinate dehydratase, and predicted to be responsible for the formation of DHS.
4.5 Microarray analysis
The aromatic amino acids phenylalanine, tyrosine and tryptophan are synthesized in E. coli via the shikimate pathway, a well-studied and widely conserved biosynthetic pathway that involves 17 enzymes, and multiple levels of regulation (Bentley, 1990; Knaggs, 1999; Pittard, 1996). When analysing similar pathways in novel microorganisms, particularly those with non-canonical enzymatic steps such as haloarchaea, a number of strategies need to be considered. At the transcriptional level one might use traditional methods for detecting gene expression changes such as Northen blotting or differential RNA display, which allow analysis of only one or few genes at a time. DNA microarrays on the other hand are a much more powerful tool for studying genome-wide differential gene expression. Microarrays allow high throughput, parallelism, speed and automation and by encompassing the whole genome it eliminates bias associated with preselecting a subset of genes believed to be involved in certain cellular event. Nonetheless, microarray experiments produce voluminous datasets which are frequently difficult to analyse and can lead to confusing hypotheses and conclusions (Dharmadi and Gonzalez, 2004).
DNA Microarrays were developed in the early nineties of the last century and have been used successfully to study gene expression in many organisms, including plants (Aharoni, 2001), pathogen-infected hosts (Diehn and Relman, 2001), humans (Kurella et al., 2001), and bacteria, such as Bacillus subtilis and E. coli (Eymann et al., 2002; Laub et al., 2000; Rohlin, 2002; Ye et al., 2000). Until 2001, no genome-wide transcriptome analysis had been performed on any archaeal species, and only one example was published using an array based on 271 ORFs from the hyperthermophilic archaeon Pyrococcus furiosus (Schut et al., 2001). In recent years, DNA microarrays of archaeal species have become an important tool for studying complex biological processes, such as understanding the regulatory network underlying the biochemical pathways for phototropy in H. salinarum NCR-1 strain (array based on 2413 ORFs (Baliga et al., 2002)). Microarrays were used as well to investigate chromosome replication in the hyperthermophilic acidophiles S. acidocaldarius and S. solfataricus using arrays based on 1,914 and 2,488 ORFs,
respectively (Lundgren et al., 2004). In 2006, the genome-wide DNA microarray (1722 ORFs) made of Methanococcus maripaludis was published, which validated proteomic data by microarray and RT-PCR (Xia et al., 2006). This was followed by a publication from Twellmeyer et al., (Twellmeyer et al., 2007)which established the first genome-wide DNA microarray of H. salinarum R1 (2774 ORFs). This array has been used to analyse the transcriptional differences between cells grown under aerobic and phototrophic conditions (Twellmeyer et al., 2007), the phosphate- dependent gene expression in H. salinarum (Wende et al., 2009), the global and specific transcriptional regulatory effects of Lrp-like proteins in H. salinarum (Schwaiger, 2010), and the AroAAs-related genes of the shikimate pathway (this study).
4.6 Objectives
The pathway leading to the biosynthesis of aromatic amino acids, known as the shikimate pathway, is well understood in both, Eukaryotes and Bacteria, where the genes, enzymes and the reactions, for each step have been intensively studied (Daugherty et al., 2001; Pittard, 1996). This pathway is critical for growth as, in addition to the biosynthesis of AroAAs, it is the source of p-aminobenzoic acid (the precursor of folic acid), and p-hydroxybenzoic acid, the precursor of the quinones, which are members of the electron transport chain. Analysis of the haloarchaeal genomes has failed to identify the first two genes in this pathway, but from studies on the euryachaeota M. jannaschii (White, 2004), a pathway in haloarchaea can be postulated. This proposed non-canonical pathway is the subject of this investigation in Halobacterium salinarum, with the aims to provide genetic and biochemical evidence for the full de novo pathway of aromatic amino acids in haloarchaea.
The specific objectives related to verifying the proposed pathway were:
1) The construction of mutants of each of the first three genes: OE1472F, OE1475F and OE1477R (figure #10A-B). Deletion of these genes would be expected to convert H. salinarum to aromatic amino acid auxotrophy.
2) Determining the phenotypes of these mutants including nutrient requirements, growth rates and their ability to import AroAAs from the medium.
3) Assessing the differential gene expression of all assigned genes for the AroAAs biosynthesis pathway using a DNA microarray of H. salinarum. 4) Expression and purification of the gene products of OE1472F, OE1477R and
OE2019F in order to examine their enzymatic activities.
5) Developing the relevant biological assays to determine the activities, kinetics and substrate specificities of the purified OE1472F, OE1477R and OE2019F proteins.