Biochemical and structural characterization of the BioZ enzyme engaged in bacterial biotin synthesis pathway

Abstract

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of β-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.

Fig. 1. Requirement of biotin for Agrobacterium growth.

a Schematic illustration of the biotin-auxotrophic strain of A. tumefaciens (ΔbioBFDA). The removal of biotin operon bioBFDA from A. tumefaciens NTL4 interrupts the biotin synthesis pathway of A. tumefaciens, and therefore interferes bacterial growth on the condition without the supplementation of exogenous biotin. b Growth rescue of the ΔbioBFDA mutant by the addition of exogenous biotin. c Unlike biotin, its precursor dethiobiotin (DTB) fails to allow the ΔbioBFDA mutant to appear on the nonpermissive growth condition without any biotin. d The growth of the ΔbioBFDA mutant on the biotin-lacking medium is restored by the cross-feeding with the wild-type strain of A. tumefaciens NTL4. e The cell-free growth culture (i.e., supernatant) of A. tumefaciens NTL4, (rather than the negative control, Klebsiella pneumoniae strain 24) cross-feeds the biotin auxotroph ER90 (ΔbioFCD) of E. coli. Here, the A. tumefaciens NTL4 was pelleted and then suspended with 1× PBS. It indicated that A. tumefaciens NTL4 secrets biotin (mostly its DTB precursor) into the growth medium/environment. The strain FYJ283 (ΔbioBFDA) appeared as an indicator strain in the biotin (DTB) bioassay (b–c), and also acted as a recipient strain in the cross-feeding experiment of A. tumefaciens NTL4 (d). The biotin/DTB bioassay was routinely performed as earlier described^,. At Agrobacterium tumefaciens, Kp24 Klebsiella pneumoniae strain 24.

Fig. 2. Phylogeny of BioZ and its paralogs.

a An unrooted tree of BioZ proteins and its putative homologs (FabH, FabB, and FabF). The phylogenetic tree was generated with the MEGA7 software using the NJ method (bootstrap: 1000 replicates). Two major clades of KAS enzymes are shown: FabF in red and FabH in orange. In contrast, FabB (shown in gray) is phylogenetically positioned as a sub-branch of FabF clade and BioZ was localized to a sub-branch of FabH clade. Therefore, we speculated that FabB originates from FabF (KAS II), and a recent ancestor of BioZ arises from FabH, the KAS III enzyme within the KAS pan-family. b Phylogenetic relationships of BioZ paralogs. Phylogeny was constructed using the MEGA7 software involving the NJ method (bootstrap: 1000 replicates, bootstrap values indicated by circle sizes). BioZ of Agrobacterium and Brucella melitensis are colored in red and blue, respectively.

Fig. 3. A role of BioZ in the FAS II-involving biotin biosynthesis.

a In vitro biosynthesis of pimeloyl-ACP and/or its precursors. Using the FAS II system, BioZ catalyzes the synthesis reaction of pimeloyl-ACP from malonyl-ACP and glutaryl-CoA (ACP). The reaction mixture was separated with conformation-sensitive urea polyacrylamide gel electrophoresis (PAGE). A representative result is given from three trials. Of note: 17.5% PAGE (pH 9.5) containing 0.5 M urea was used here. The two controls (C4-ACP and C6-ACP) served as standards/markers for this conformation-sensitive urea gel. C7-ACP denotes four species of ACP attached with an acyl seven-carbon fatty acyl chain, namely, 5-keto-pimeloyl-ACP, 5-hydroxyl-pimeloyl-ACP, enoyl-pimeloyl-ACP, and pimeloyl-ACP. b MS/MS identification of 5-keto-pimeloyl-ACP, a primary product from the BioZ reaction coupled with FAS II, using glutaryl-CoA and malonyl-ACP as substrates. The use of MS/MS allowed us to detect the presence of four C7-ACP species in the above reaction system. As for an initial product of BioZ reaction, 5-keto-pimeloyl ACP, a 15-residue peptide fragment of interest is given. The C7-fatty acyl modification with high reliability is localized on the conserved Serine 36 of ACP. The two peaks of peptide fragments indicated with pink arrows were used to determine C7 acyl modification. The resultant mass was 498.1533, which is close to the theoretical mass (498.143) of Ppan-linked keto-pimeloyl moiety. Of note, it might be not as stable as pimelic acid. c Cartoon illustration of the pimeloyl-ACP structure. This was generated from the complex structure of methyl-pimeloyl-ACP and BioH (PDB: 4ETW) with appropriate modifications. d A scheme for BioZ bypassing the canonical early steps of biotin synthesis. Unlike the paradigm “BioC-BioH” mechanism of biotin synthesis (above the dashed line), the BioZ reaction bypasses the earlier steps of “BioC-BioH” in biotin biosynthesis (below the dashed line). Recently, glutaryl-CoA is determined to physiologically arise from lysine catabolism in Agrobacterium species. Designations: C4-ACP butanoyl-ACP, C6-ACP hexanoyl-ACP, Mal-ACP malonyl-ACP, Glu-ACP glutaryl-ACP, Pim-ACP pimeloyl-ACP.

Fig. 4. Structural comparison of four FAS-type enzymes.

a Ribbon representation of the E. coli FabB structure (PDB: 1G5X). b Ribbon structure of the E. coli FabF (PDB: 2GFW). c Ribbon representation of the E. coli FabH structure (PDB: 1HN9). d Ribbon illustration of the A. tumefaciens BioZ structure (PDB: 6KUE). The lid domains in EcFabB (a), EcFabF (b), and EcFabH (c) are separately colored in green, cyan, and yellow, respectively. Whereas, the core domains are colored in silver–gray. As for AtBioZ, the lid domain is shown in red, and its core domain appears in blue (d). The three catalytic triad residues are indicated with pink letters.

Fig. 5. Structural and functional analyses of the BioZ catalytic triad.

a Structural snapshot of the catalytic triad (C163, H298, and H333) of EcFabB. b Structural analysis of the catalytic triad (C164, H304, and H341) of EcFabF. c Structural presentation of the catalytic triad (C112, H244, and N274) of EcFabH. d Structural illustration of the catalytic triad (C115, H255, and N285) of AtBioZ. The figures of structures were generated using the PyMol software. e Functional assays of BioZ and its catalytic triad mutants using the ΔbioH biotin-auxotrophic strain. The tested strains were grown on nonpermissive M9 minimal media lacking biotin. Growth curves were generated from three independent experiments and displayed in an average ± standard deviation (SD).

Fig. 6. Structural and functional analysis of BioZ binding to malonyl-ACP.

Surface structure (a) and ribbon illustration (c) of the FabB dimer crosslinked with ACP. It was generated with PyMol using crystal structure of FabB-ACP (PDB: 5KOF). Surface structure (b) and ribbon illustration (d) of the dimeric BioZ complexed with malonyl-ACP. e Use of isothermal titration calorimetry (ITC) to probe the interaction between malonyl-ACP and BioZ. A representative result of ITC is shown, and the resultant stoichiometry values (N and Kd) from three independent experiments are given in an average ± SD. The putative binding mode of BioZ to its substrate malonyl-ACP (and/or glutaryl-ACP) was generated through structural superposition. The stoichiometry (malonyl-ACP: BioZ) is 1:1, in that the molar ratio was measured to be ~0.996. The parameter of binding affinity (Kd) is 24.133 ± 0.602 μM.

Fig. 7. Structural insights into the recognition of malonyl-ACP by the BioZ enzyme.

a Structural display of the ACP moiety of acyl-ACP substrate bound to the paradigm enzyme of FAS I, the E. coli FabB. b An enlarged view of the ACP-interacting interface of FabB that is constituted by six positively-charged residues. The image is generated through a counter-clockwise 90° rotation of the rectangle-dashed lined region (a). c Binding model of glutaryl-ACP to the positively-charged surface of AtBioZ. ACP is given in ribbon structure colored gray, which comprises four α-helices (helix-1 to helix-4). The interface of FabB (a) [and/or BioZ (c)] interacting with ACP group is illustrated in the surface electrostatic structure. The blue denotes positive charge, whereas the red refers to negative charge. d Visualization of the putative ACP-binding domain of BioZ. Presumably, it contains four basic residues, namely R39, R153, R221, and R260. This image is given through the counter-clockwise 90° rotation of the square-dashed lined region (c). e Growth curve-based assay to probe the role of the putative four basic residues-comprising, ACP-binding interface in the BioZ function. The strains used here were listed in Supplementary Table 1, and the growth curve was plotted as described in Fig. 5e. A representative result was given.

Fig. 8. Biochemical evidence that BioZ binds to its substrate glutaryl-CoA.

a Molecular docking of BioZ with glutaryl-CoA. b Structural snapshot of the glutaryl-CoA substrate-loading tunnel within BioZ enzyme. c Use of ITC to measure the binding of glutaryl-CoA to BioZ protein. A representative ITC result from three independent experiments is displayed. Thus, the stoichiometry values (N and Kd) are expressed in an average ± SD. The stoichiometry of glutaryl-CoA binding to BioZ enzyme is proposed to be 1:1, in that the molar ratio was measured to be around 0.8. The parameter of binding affinity (Kd) is 2.64 ± 0.23 μM.

Fig. 9. Functional dissection of the glutaryl-CoA substrate-loading tunnel within the BioZ enzyme.

a The proposed critical residues of BioZ contributing to the neutralization of free carboxyl group of glutaryl-CoA. Three residues that might neutralize the charge of the free carboxyl group from glutaryl-CoA is determined by molecular docking to localize at the distant end of substrate-loading pocket (Fig. 8a, b). Namely, they are Serine 84 (S84), Arginine 147 (R147), and Serine 287 (S287), respectively (a). b The three possible neutralizing residues (S84, R147, and S287) are conserved across all the BioZ homologs from different α-proteobacteria. Sequence logo was generated using the server WebLogo (http://weblogo.berkeley.edu/logo.cgi). c Use of site-directed mutagenesis to assay the varying roles of the three putative charge-neutralizing residues in BioZ activity. d Growth curves of the ΔbioH strains carrying either the wild-type bioZ or its point-mutants. Derivatives of the ΔbioH biotin-auxotrophic strain (Supplementary Table 1) were assayed on the nonpermissive growth condition of M9 minimal media without any biotin. The addition of 0.2% arabinose induced the expression of pBAD24-borne bioZ (and/or its point-mutants).