Biochemical and structural characterization of the BioZ enzyme engaged in bacterial biotin synthesis pathway.
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ABSTRACT:
SUBMITTER: Zhang S
PROVIDER: S-EPMC8024396 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag") act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.
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Project description:Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.
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Project description: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.
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Project description:Biotin is an indispensable cofactor in the three domains of life. The unusual virulence factor BioJ of Francisella catalyzes the formation of pimeloyl-ACP, an intermediate in biotin synthesis. Here, we report the 1.58 Å crystal structure of BioJ, the enzymatic activity of which is determined with the in vitro reconstituted reaction and biotin bioassay in vivo. Unlike the paradigm BioH, BioJ displays an atypical ?/?-hydrolase fold. A structurally conserved catalytic triad (S151, D248, and H278) of BioJ is functionally defined. A proposed model for BioJ catalysis involves two basic residues-rich cavities, of which cavity-1, rather than cavity-2, binds to the ACP moiety of its physiological substrate, pimeloyl-ACP methyl ester. In summary, this finding provides molecular insights into the BioJ gatekeeper of biotin synthesis.
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Project description:There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).
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