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

Sitao Zhang,Youjun Feng,Wenhui Wei,Yuling Liao,Hongxin Guan,Huimin Zhang,Yongchang Xu,Bachar H Hassan,Tao Cui,Songying Ouyang,Jun Li,Xu Jia

doi:10.1038/s41467-021-22360-4

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.

Highlights

Biotin is an essential micro-nutrient across the three domains of life
The bioBFDA operon of A. tumefaciens encodes four enzymes responsible for the latter steps of biotin synthesis, assuring the physiological requirement of biotin (Fig. 1a). The removal of this operon impairs its capability of biotin synthesis, giving the biotinauxotrophic strain ΔbioBFDA (Fig. 1a)
(i) the wild-type strain of A. tumefaciens, NTL4 is a DTB/biotin producer, and in the ΔbioBFDA mutant acts as a recipient strain; (ii) cell-free bacterial supernatants (A. tumefaciens and Klebsiella pneumoniae Kp24 strain) serve as the DTB/biotin donors, whereas the ER90 (ΔbioFCD) strain of E. coli functions as a DTB/biotin sensor

Summary

Introduction

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. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5’-ketopimeloyl 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. Biotin synthesis proceeds in two stages: (i) the generation of pimelate moiety, an atypical α, ω-dicarboxylic acid of sevencarbons; and (ii) the assembly of a fused heterocyclic rings of biotin[16,17,18] The latter steps are highly conserved in that it consistently proceeds via four successive reactions catalyzed by four unique enzymes[2]. This increases the diversity of demethylases in the context of biotin synthesis

Methods

Results

Conclusion