Abstract
The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.
Highlights
Each subunit activates the other subunit 30 –100-fold in the reaction that that subunit can perform alone [1, 2]
In this study we have assumed that the amino-terminal segments of the TrpA polypeptides of E. coli and S. typhimurium are equivalent functionally and that we may interpret our results on the basis of the three-dimensional structure of the Tryptophan synthetase (TSase) of S. typhimurium [5]
A hybrid TrpA containing the first 60 residues of S. typhimurium TrpA and the remaining 208 residues of E. coli TrpA was functionally indistinguishable from its parental proteins [24]
Summary
Each subunit activates the other subunit 30 –100-fold in the reaction that that subunit can perform alone [1, 2]. Comparison of some of these sequences and predictions of likely secondary structures led to the initial suggestion that the TrpA subunit was a member of the class of 8-fold ␣/ barrel polypeptides [13] This prediction was validated and extended by the x-ray crystallographic determination of the structure of the tetrameric enzyme from Salmonella typhimurium [5]. The three-dimensional structure of the enzyme complex from S. typhimurium reveals that the respective amino and carboxyl termini of the TrpA and TrpB polypeptide chains are spatially separated, fusion of the two polypeptides, in either order, would require a moderately long peptide connector if the natural spatial relationships within and between the two subunits were to be retained [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
