Directed evolution of a (beta alpha)8-barrel enzyme to catalyze related reactions in two different metabolic pathways.
Enzymes participating in different metabolic pathways often have similar catalytic mechanisms and structures, suggesting their evolution from a common ancestral precursor enzyme. We sought to create a precursor-like enzyme for N'-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (ProFAR) isomerase (HisA; EC ) and phosphoribosylanthranilate (PRA) isomerase (TrpF; EC ), which catalyze similar reactions in the biosynthesis of the amino acids histidine and tryptophan and have a similar (betaalpha)(8)-barrel structure. Using random mutagenesis and selection, we generated several HisA variants that catalyze the TrpF reaction both in vivo and in vitro, and one of these variants retained significant HisA activity. A more detailed analysis revealed that a single amino acid exchange could establish TrpF activity on the HisA scaffold. These findings suggest that HisA and TrpF may have evolved from an ancestral enzyme of broader substrate specificity and underscore that (betaalpha)(8)-barrel enzymes are very suitable for the design of new catalytic activities.
Jürgens
C
C
Abteilung Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Strom
A
A
Wegener
D
D
Hettwer
S
S
Wilmanns
M
M
Sterner
R
R
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C079656:Phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase; C029865:phosphoribosylanthranilate isomerase