.
Angewandte
Communications
Enzymes
Conversion of Anthranilate Synthase into Isochorismate Synthase:
Implications for the Evolution of Chorismate-Utilizing Enzymes
Maximilian G. Plach, Patrick Lçffler, Rainer Merkl, and Reinhard Sterner*
Abstract: Chorismate-utilizing enzymes play a vital role in the
biosynthesis of metabolites in plants as well as free-living and
infectious microorganisms. Among these enzymes are the
homologous primary metabolic anthranilate synthase (AS)
and secondary metabolic isochorismate synthase (ICS). Both
catalyze mechanistically related reactions by using ammonia
and water as nucleophiles, respectively. We report that the
nucleophile specificity of AS can be extended from ammonia to
water by just two amino acid exchanges in a channel leading to
the active site. The observed ICS/AS bifunctionality demon-
strates that a secondary metabolic enzyme can readily evolve
from a primary metabolic enzyme without requiring an initial
gene duplication event. In a general sense, these findings add to
our understanding how nature has used the structurally
predetermined features of enzyme superfamilies to evolve
new reactions.
contribute to nucleophile specificity in MST enzymes and by
subsequently establishing ICS activity on an AS scaffold.
The AS from Salmonella typhimurium (stAS) is a hetero-
tetramer comprising two synthase and two glutaminase
subunits (stTrpE and stTrpG, respectively).[4] Glutamine is
hydrolyzed in the active site of stTrpG to yield nascent
ammonia, which is subsequently channeled to the active site
of stTrpE.[4,5] To identify the residues of stTrpE that are
involved in this channeling and that may therefore come into
contact with the ammonia nucleophile, we applied
MOLE 2.0, a program for analyzing macromolecular chan-
nels.[6] We identified a 30 -long channel connecting the
active sites in stAS (Figure 2a). This channel is similar to the
channel observed in the crystal structure of the homologous
aminodeoxyisochorismate (ADIC) synthase PhzE.[7] As the
channel approaches the CH ligand, it is predominantly shaped
by three residues: Gln263 in b-strand 11 as well as Met364 and
Leu365 in a-helix 12. To estimate whether the nature of these
residues correlates with nucleophile specificity in AMEs and
WMEs, we computed individual multiple sequence align-
ments (MSAs) of ADCS, AS, ICS, and SS. Notably, these
sequences formed four distinct subtrees in a phylogenetic
analysis (Figure S1 in the Supporting Information), which
makes them representative for their MST groups. Further-
more, all of the MSAs contained sequences of at least four
major phyla. The resulting sequence logos of b-strand 11 and
a-helix 12 (Figure 2b) confirmed the strict conservation of
Gln263 in AS and of Lys at the corresponding position in ICS
and SS, which has been noted previously.[2] It was further
shown that this Lys acts as a catalytic base for water activation
in ICS and SS.[2,8] In a number of ADCS sequences, Gln263 is
substituted by Glu. Residues 364 and 365 are conserved to
a large extent within AMEs and WMEs, but clearly differ
between the two groups. In AMEs, Met364 is strictly
conserved, as are Leu365 in AS and Ile365 in ADCS. In
WMEs however, several hydrophobic residues (Leu, Ile, Phe,
Val) are abundant at position 364, as are Val in ICS and Ser in
SS at position 365. Other positions were not considered since
they are either conserved throughout all four MST groups, not
conserved within AMEs or WMEs, or not involved in forming
the nucleophile channel in stAS.
C
horismate (CH) is a central metabolic branch-point
molecule and the common precursor of essential primary
(folate, tryptophan) and important secondary (menaqui-
nones, siderophores, antibiotics) metabolites that are vital
for plants as well as free living and infectious microorgan-
isms[1] (Figure 1). The CH-related pathways are therefore
notable targets for antimicrobials and herbicides. The
enzymes catalyzing the committed steps of these pathways
share a common fold and use similar reaction mechanisms.
Presumably, they originated from a common ancestor and
have therefore been grouped together as the MST (menaqui-
none, siderophores, tryptophan) superfamily.[2] Within this
superfamily, the primary metabolic anthranilate and amino-
deoxychorismate synthases (AS, ADCS) employ ammonia as
a nucleophile to form aminated chorismate derivatives,
whereas the secondary metabolic isochorismate and salicylate
synthases (ICS, SS) use water as a nucleophile to hydroxylate
chorismate (Figure 1). These two subfamilies are hereafter
termed ammonia-utilizing and water-utilizing MST enzymes,
respectively (AMEs, WMEs). Based on the assumption that
each enzyme of secondary metabolism stems from an enzyme
of primary metabolism,[3] the subdivision of the MST super-
family suggests that a transition of nucleophile specificity
from ammonia to water underlay the evolution of WMEs
(ICS, SS) from AME (AS, ADCS) ancestors. We retraced this
putative evolutionary path by identifying the residues that
Based on this analysis, we attempted to shift the nucle-
ophile specificity of stAS from ammonia to water by mutating
residues 263, 364, and 365 of the stTrpE subunit. For this
purpose, 16 variants were generated and assayed by HPLC for
the formation of reaction products starting from CH. The
WME-typical catalytic Lys replaced the wild-type Gln263 in
all of the variants and was combined with the different
residues 364/365 found in WMEs (Table S1). The variants are
hereafter denoted by their residues 263–364–365 combination
[*] M. G. Plach, P. Lçffler, Prof. Dr. R. Merkl, Prof. Dr. R. Sterner
Institut für Biophysik und physikalische Biochemie
Universität Regensburg, 93040 Regensburg (Germany)
E-mail: Reinhard.Sterner@ur.de
Supporting information for this article is available on the WWW
11270
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 11270 –11274