W Mazhawidza, M Somuah, N Rajendran
arthrobacter nicotianae, glutamate synthase, phylogenetics
W Mazhawidza, M Somuah, N Rajendran. Molecular Probing and Phylogenetic Analysis of Glutamate synthase of Arthrobacter nicotianae.. The Internet Journal of Microbiology. 2008 Volume 7 Number 2.
Glutamate synthase (GltS) is an enzyme complex of NADP, iron, sulfur and FMN, that catalyses the reversible oxidoreductive biosynthesis of L-glutamate from 2-oxoglutarate and L-glutamine using ammonia (1). This reaction occurs in bacterial, yeast and as well as plant cells. This enzymatic complex interaction plays a central in amino acid metabolism as well as maintaining control of ammonia levels (1). Other names for the enzyme are glutamine-ketoglutaric aminotransferase and glutamine amide-2-oxoglutarate aminotransferase. The mechanism of action involves NADP or FMN as a cofactor. The enzyme can also utilize other cofactors like FAD, iron and sulfur as alternative cofactors. It is on the bases of these cofactors that Glt(S) is classified into three groups, the NADPH-dependent in bacteria, ferredoxin-dependent in plants and NAD (P)H-dependent in other lower animals. Specifically, this group of enzymes acts on CH-NH2 as donor groups and the cofactors as the acceptors.
Glutamate is very central to the metabolism of amino acids as its carbon chain or amide group can be directed into different biosynthetic pathways. Whilst the mechanism of L-glutamine biosynthesis in lower organisms and plants is mediated by glutamate synthase, it is part of a dual pathway that is dependent on the abundance energy in the form of ATP, the levels of ammonia and glutamine which is convertible back to glutamate(2). Specifically E. coli has a dual pathway whose activities are inhibited or activated by ATP and ammonia (3, 4). These alternate pathways allow the bacteria to regulate the levels of glutamate, glutamine and ultimately assimilate ammonia. The same enzyme mechanisms are also used by other bacterium like
Previous cross species phylogenetic analysis revealed unique phenomena about glutamate synthase and homologous enzymes in prokaryotes (7). Lateral gene transfer, was suggested by the scattered ness of glutamate synthase and homologous genes (7).
Given the critical role this enzyme plays in amino acid metabolism it is important to understand its evolutionary position across any related species particularly the large community of soil inhabiting bacteria. In our study, we attempted to perform phylogenetic analysis of glutamate synthase in soil isolated
Materials and Methods
Bacterial Culture methods and DNA isolation:
Nutrient Broth culture (10mL) of
CER1 and EMT1 primers (8) were used in this study. Primers were synthesized at Integrated DNA Technologies, Coralville, IA, USA. The PCR reactions were carried out as standardized earlier(9). Thermocycler (Eppendorf Mastercycler personal, Eppendorf) was used to cycle the PCR. The following conditions: 95 ºC (5 min), 95 ºC (1 min), 55 ºC (1 min), and 72 ºC (3 min) for 30 cycles were adopted.
Subcloning and DNA sequencing:
Amplicons obtained from PCR were sub cloned in
Sequence analysis, construction of phylogenetic tree and 3D analysis:
After confirmation of the DNA sequence using NCBI vector contamination software (vecscreen), further computational analysis was conducted. Homologous nucleotide and or amino acid sequence search was performed using NCBI blast search of protein databases (http://www.ncbi.nlm.nih.gov/BLAST/). The newly determined Glutamate Synthase sequence was deposited in GenBank and an accession number FJ979920 was obtained using the BankIT:GenBank (www.ncbi.nlm.nih.gov/BankIT/) submission program. To confirm the specificity signature sequence conserved amino acid residue search was performed during multiple sequence alignment. MegAlign software was then used to generate the phylogenetic tree.
To determine the domain structure of the clone, Cn3D 4.1 (NCBI) software was used. The newly cloned sequence was compared to 3D domain model constructed from multiple sequence alignment of many peptides in the same class. Domain alignment was performed to demonstrate common and consensus regions. Images were exported from Cn3D software as JPEG.
Nucleotide primers (8) designed from conserved domains of nonribosomal peptide synthetase were used in PCR to amplify corresponding regions on Arthrobacter nicotianae genomic DNA. As shown in Figure 1, we were able to amplify a
Seeking the consensus sequences, homologous amino acid residues and 3D domains
In an effort to find a putative identity of the gene partial sequence, we analyzed the amplicon sequence by multiple sequence alignment using multiple sequence alignment software, MegAlign, DNAstar (Fig 2). Alignment was performed with potential matches obtained from NCBI blast search of microbe protein databases. Using ClustalW, we revealed the presents of conserved amino acid residues shown in figure 2. A thorough search of the conserved domains revealed that the newly cloned sequence matched in part to the TIM barrel of the GltS as well as having FMN binding consensus amino acid residues (Fig. 2). A comparison of the TIM barrel primary structure and our
Tertiary structure analysis of the newly sequenced GltS was performed using Cn3D 4.1 software (NCBI) (13). Conserved amino acid residues of the structural alpha and beta sheets that form the TIM barrel are present in
Phylogenetic tree of partial glutamate synthase sequence cloned from
In our study we employed a PCR approach in revealing part of the genome of a newly isolated and identified soil bacterium. As noted before
GltS is a large protein with many functional domains(14). What we accomplished is cloning the middle section of the gene with structural function as well as the binding of cofactors. As shown in figure 3, the GltS cloned has amino acid residues critical for FMN binding. Missing from the clone are amino acid residues that form the substrate binding site as well as ferrodoxin cluster (14). To our knowledge this is the first time GltS has been cloned in
GltS and phylogenetic analysis
Glutamate synthase is phylogenetically interesting as well as metabolically pivotal to the biology of bacteria and other organisms (15-20). Previous phylogenetic analysis revealed that GltS had undergone lateral gene transfer from bacteria to a common ancestor of animals, fungi, and plants (21). Our analysis demonstrates a close relationship between strains of the same type of bacteria as expected. Unique is that
It is important to highlight a few bacteria that matched with the newly cloned GltS. In
The availability and use of software like Cn3D 4.1 has potential to positively impact analysis and discovery of life science data like we have not seen before (13, 29-33). By importing the newly cloned GltS sequence into the software we were able to align the primary structure as well as determine conserved domains and view them in their putative three dimensional forms. This approach to analysis of genomic data as we have seen has the potential to identify and rapidly unveil the metabolism as well as the phylogentic position of the organism.
We revealed the presence of glutamate synthase in
This work was supported by National Science Foundation, USA (Grant # MCB-0639356 to Narayanan Rajendran). Authors thank ASM/JGI(DOE) and Drs. Brad Goodner, Cheryl Kerfeld, Kathleen Scott and Sam Donovan of ASM Spring-2009 Bioinformatics Institute for sequence alignment, phylogenetic and bioinformatics training.