We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side-chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the alpha/beta-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.
Title: Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH Flores H, Lin S, Contreras-Ferrat G, Cronan JE, Morett E Ref: Protein Engineering Des Sel, 25:387, 2012 : PubMed
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.
Title: Biotin synthesis begins by hijacking the fatty acid synthetic pathway. Lin S, Hanson RE, Cronan JE Ref: Nat Chemical Biology, 6:682, 2010 : PubMed
Although biotin is an essential enzyme cofactor found in all three domains of life, our knowledge of its biosynthesis remains fragmentary. Most of the carbon atoms of biotin are derived from pimelic acid, a seven-carbon dicarboxylic acid, but the mechanism whereby this intermediate is assembled remains unknown. Genetic analysis in Escherichia coli identified only two genes of unknown function required for pimelate synthesis, bioC and bioH. We report in vivo and in vitro evidence that the pimeloyl moiety is synthesized by a modified fatty acid synthetic pathway in which the omega-carboxyl group of a malonyl-thioester is methylated by BioC, which allows recognition of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl-acyl carrier protein (ACP) methyl ester, which is hydrolyzed to pimeloyl-ACP and methanol by BioH.
