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Thursday, 22 February 2018
Tryptophan Biosynthesis PDF Print
*       Homology Modeling of Anthranilate Synthase Subunits of Chromobacterium violaceum
Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite with pharmacological interest. Genome analysis of this bacterium showed that the genes trpA-F and pabA-B encode four enzymes that catalyze five reactions of the tryptophan biosynthesis pathway. The first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzymatic complex. The structure and organization of AS biosynthesis genes of C. violaceum were analyzed by using bioinformatics tools available on the Web. We have shown by calculating molecular weights that AS in C. violaceum is composed by a (trpE) and b (pabA) subunits. This is in agreement with values determined experimentally. The catalytic and regulatory sites of the AS subunits were identified. TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit shows an allosteric site. The protein models for TrpE and PabA subunits built from homology with the Salmonella typhimurium AS enzyme, chains A (1I1QA) and B (1I1QB), showed high similarities, respectively 98.5% and 96.8% of amino acids in favorable energy regions.


*       Modeling and Dynamic Simulation of trp Operon in Bacillus subtilis
Seven genes encode the enzymes of Bacillus subtilis L-tryptophan (Trp) biosynthesis pathway. Six of these seven genes are organized as a trpEDCFBA operon that is a suboperon within histidine and aromatic amino acid supraoperon. Expression of the trp operon is negatively regulated by the intracellular tryptophan level through tryptophan-activated TRAP protein (trp RNA-biding attenuation protein). TRAP contains 11 identical subunits and is activated by the cooperative binding of Trp molecules that bind between TRAP adjacent subunits, activating it to bind to the trp leader transcript.

It is known that the transcription of the trp operon starts 203 nucleotides (trp leader RNA) upstream of the trpE start codon. The trp leader RNA is able to form two secondary structures that are known as antiterminator and terminator. The formation of both structures is mutually exclusive, because there are four base pairs shared by them downstream of the antiterminator. The activated TRAP binds to trp leader RNA in a region that contains 11 triplets, seven GAG and four UAG. Doing so it prevents antiterminator formation and induces formation of terminator hairpin and, consequently, halting expression of the trp operon in the trp leader RNA. Under conditions of limiting tryptophan, TRAP does not bind to RNA and an alternative antiterminator structure forms, leading to read-through of the operon and expression of the trp genes.

The trpE translation control also is carried by formation of trp leader RNA hairpin. TRAP binding to trp read-through RNA transcripts that have escaped from attenuation promotes formation of another RNA secondary structure that sequesters the trpE ribosome-binding site thus inhibiting translation of trpE. In the absence of TRAP, the leader transcript forms a structure in which trpE Shine-Dalgarno is free to bind to ribosome and initiates its translatio. In addition to the regulations seen above, the anthranilate synthase (AS) enzyme activity, the first Trp pathway enzyme, is inhibited by Trp.

We developed a mathematical dynamic model as an ODE system with variables M, E and W representing, respectively, mRNA, AS enzyme and Trp expression levels. We consider all kind of regulation described above.

With the parameter values given in the literature we simulated the following derepression experiment involving the anthranilate synthase enzyme.

Let bacteria grow exponentially in a Trp-rich medium; then, at time zero, switch them to a medium without Trp and thereafter the AS activity is measured. In the simulation of derepression experiments the following set of initial conditions have been employed: M=0, E=0 and W=10WSS (where, WSS is the steady state tryptophan concentration). This set of initial values corresponds to a state in which the trp operon is repressed due to a high intracellular tryptophan level. The model equations were then numerically solved and the simulation results compared with the experimental data, given good agreement.

In both data there is an overshoot indicating that the tryptophan pool size does not reach physiologically repressing levels until after the second cell division (250-300 min). In the simulated results the AS activity increases more rapidly than those obtained experimentally. This happens due to the chosen regulation functions and their parameters that behave, in general, as a single step function. In that way regulation is released only in a short Trp concentration range. In transcription and translation processes there are time delays involved not taken into account. Also our model did not consider the AT protein. AT protein inhibits the TRAP ability to bind to trp leader RNA, therefore promoting trp operon expression. Future work should address those questions.
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