Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH

Abstract

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.

Expression and Activity of the BioH Esterase of Biotin Synthesis is Independent of Genome Context

“…2a). Expression of the P. aeruginosa in an E. coli ∆bioH strain resulted in robust growth in biotin-free medium as expected from prior studies with various nonorthologous esterases 24, 25, 30, 31 (data not shown). Alignments of the two BioH proteins argue that the two enzymes share the same esterase catalytic triad (Ser-His-Asp) (Fig.…”

“…The three BioH proteins with demonstrated pimeloyl-ACP methyl ester cleavage activity are given in large bold type. The PA3859 protein is included as the outlier since it is a P. aeruginosa α/β-hydrolase known to lack pimeloyl-ACP methyl ester cleavage activity 31 . The blue dotted box with the exception of PA3859 denotes the operon-encoded BioH proteins.…”

“…In this regard it is interesting that evolution of a P. aeruginosa carboxylesterase (PA3859) of unknown function to gain BioH activity required only simple amino acid substitutions 31 . Moreover only when high in vitro activity against pimeloyl-ACP methyl ester was attained did the mutant PA3859 proteins support growth at rates similar to that of E. coli BioH 31 .…”

“…In this regard it is interesting that evolution of a P. aeruginosa carboxylesterase (PA3859) of unknown function to gain BioH activity required only simple amino acid substitutions 31 . Moreover only when high in vitro activity against pimeloyl-ACP methyl ester was attained did the mutant PA3859 proteins support growth at rates similar to that of E. coli BioH 31 . A recent study of the enzyme encoded by Haemophilus influenzae bioG , the gene most often found in place of bioH immediately upstream of bioC , encodes an enzyme of activity comparable to that of E. coli BioH 42 .…”

“…Moreover the P. aeruginosa and Bacillus cereus proteins showed only 21% identity. Indeed when the BioH sequences were submitted to Clustal Omega alignment and the results analyzed according to Kimura 31, 39, 40 , the freestanding and operon-encoded BioH proteins fell cleanly into two clades. Moreover, the same clades were found when two other analysis approaches, Uncorrected Pairwise Distance and Scoredist 46 were applied.…”