A new suite of tnaA mutants suggests that Escherichia coli tryptophanase is regulated by intracellular sequestration and by occlusion of its active site

Table 1 Mutations that do not affect TnaA localization or enzyme activity (Category 1)

Variant	Residue(s) changed to alanine	Residue location ^a	TnaA-sfGFP localization ^b	D1D3-sfGFP Localization ^c	Indole ^d (%)
A1	K5	D1 surface	Wild type	N/A^e	96.5
A3	E17, K20, R21	D1 surface	Wild type	Wild type	101.3
A4	T23, R24, Y26	D1 surface	Wild type	Wild type	99.5
A6	K33, S34	D1 surface	Wild type	Wild type	102.7
A10	K115	D2 surface	Wild type	N/A	93.0
A11	K156	D2 surface	Wild type	N/A	98.7
A12	K239	D2 surface	Wild type	N/A	100.1
A14	N327, D329	D3 surface	Wild type	Wild type	96.8
A15	Q339, Y340, D343	D3 surface	Wild type	Wild type	99.6
A16	E346, E347	D3 surface	Wild type	Wild type	93.1
A18	D363, K366	D3 surface	Wild type	Wild type	97.7
A19	H370, D374, Q375	D3 surface	Wild type	Wild type	99.2
A21b	R403	D3 loop	Wild type	N/A	103.3
A21c	D404	D3 loop	Wild type	N/A	105.4
A22	K406, K409, Q410	D3 loop	Wild type	Wild type	100.9
A26	E437, K440, H441	D3 surface	Wild type	Wild type	92.7
A27	K443, E444, N445, N448	D3 surface	Wild type	Wild type	103.4
A28	K450	D3 surface	Wild type	N/A	97.9
A30	K459	D3 surface	Wild type	N/A	102.9
A31c	T465	D3 surface	Wild type	N/A	72.7
A32	K467, K469, E470	D3 surface	Wild type	Wild type	92.4
A33	K5, K115, K156, K239, K450, K459	Surface	Wild type	Wild type	82.5

^aResidue location is based on the crystal structure of TnaA (PDB ID 2OQX). Pocket, the active site cavity; loop, the loop comprised of aa 398 to 416 at the edge of the catalytic pocket.
^bWild type: spherical focus at midcell or one of the poles at mid-to-late exponential phase, becoming more diffuse at stationary phase as wild type TnaA.
^cWild type: spherical focus at midcell or one of the poles at mid-to-late exponential phase. D1D3 constructs were mostly degraded at stationary phase.
^dThe amount of indole produced by each TnaA-sfGFP variant compared to that produced by wild type TnaA in LB at an OD₆₀₀ of 1.5. Values are reported as the percentage of wild type, and are the average of two independent cultures. Growth rates of all cells carrying wild type TnaA or any of its variants were nearly identical.
^eN/A indicates that the variant was not constructed or that data was not available.

ISSN: 1471-2180