T. Stachelhaus et Ct. Walsh, Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase, BIOCHEM, 39(19), 2000, pp. 5775-5787
The epimerase (E) domain of the three-domain (ATE) initiation module of Bac
illus brevis gramicidin S synthetase equilibrates the C alpha configuration
of the phenylalanyl moiety presented as Phe-S-4'-phosphopantetheine-modifi
ed (Ppant) acyl enzyme. Mutants at 22 residues of this E domain that are co
nserved across the approximately 450 residue E domains of nonribosomal pept
ide synthetases were constructed, and the PheATE* derivatives expressed in
Escherichia coli as C-terminal His tag fusions and then purified and assaye
d for three activities: (1) the L-Phe C alpha-[H-3] exchange to solvent, (2
) the rate of approach to D-Phe/L-Phe-S-Ppant acyl enzyme equilibrium from
either L- or D-Phe, and (3) the rate of Phe-Pro dipeptidyl-S-Ppant enzyme f
ormation with the downstream ProCAT module. We found that for wild-type Phe
ATE epimerization is much faster than subsequent condensation, leading to a
1.9:1 ratio of D-Phe-S-Ppant/L-Phe-S-Ppant acyl enzyme. Only D-Phe is then
transferred to yield D-Phe-L-Pro-S-Ppant ProCAT acyl enzyme. Among the mut
ants generated, three PheATE" constructs, H753A, D757S, and Y976A, showed n
o detectable C alpha-H-3 washout, while E892A and R896A were among a larger
set partially impaired. All these mutants were dramatically impaired in ap
proach to D-Phe/L-Phe-S-Ppant equilibrium from either D- or L-Phe, while an
other construct, D767S, was asymmetrically impaired only for D-to-L-Phe dir
ection, In the D-Phe-L-Pro dipeptidyl-S-Ppant condensation assay, the H753A
and E842A forms of PheATE" were only slightly active from L-Phe but unimpa
ired from D-Phe; N975A epimerizes faster than Y976A from L-Phe. When the ch
irality of the Phe-Pro-diketopiperazine released product was analyzed the D
,L/L,L ratio from wild-type PheATE and ProCAT was 98:2, From E892A and N975
A it was comparably 95:5 and 92:8, but H753A and Y976A yielded 56% of the L
,L-product, reflecting a gain of function to transfer L-Phe. The 98:2 prefe
rence of wild-type PheATE for D-Phe transfer reflects the kinetically contr
olled stereopreference of the condensation (C) domain of ProCAT for the D-P
he-S-Ppant donor substrate. It may be that other NRPS C domains immediately
downstream of E domains will likewise be D-selective.