Crystal structure and acetylation of BioQ suggests a novel regulatory switch for biotin biosynthesis in Mycobacterium smegmatis

Abstract

Biotin (vitamin B7), a sulfur-containing fatty acid derivative, is a nutritional virulence factor in certain mycobacterial species. Tight regulation of biotin biosynthesis is important because production of biotin is an energetically expensive process requiring 15-20 equivalents of ATP. The Escherichia coli bifunctional BirA is a prototypical biotin regulatory system. In contrast, mycobacterial BirA is an unusual biotin protein ligase without DNA-binding domain. Recently, we established a novel two-protein paradigm of BioQ-BirA. However, structural and molecular mechanism for BioQ is poorly understood. Here, we report crystal structure of the M. smegmatis BioQ at 1.9 Å resolution. Structure-guided functional mapping defined a seven residues-requiring motif for DNA-binding activity. Western blot and MALDI-TOF MS allowed us to unexpectedly discover that the K47 acetylation activates crosstalking of BioQ to its cognate DNA. More intriguingly, excess of biotin augments the acetylation status of BioQ in M. smegmatis. It seems likely that BioQ acetylation proceeds via a non-enzymatic mechanism. Mutation of this acetylation site K47 in BioQ significantly impairs its regulatory role in vivo. This explains in part (if not all) why BioQ has no detectable requirement of the presumable bio-5'-AMP effecter, which is a well-known ligand for the paradigm E. coli BirA regulator system. Unlike the scenario seen with E. coli carrying a single biotinylated protein, AccB, genome-wide search and Streptavidin blot revealed that no less than seven proteins require the rare post-translational modification, biotinylation in M. smegmatis, validating its physiological demand for biotin at relatively high level. Taken together, our finding defines a novel biotin regulatory machinery by BioQ, posing a possibility that development of new antibiotics targets biotin, the limited nutritional virulence factor in certain pathogenic mycobacterial species.

Molecular Basis of BioJ, a Unique Gatekeeper in Bacterial Biotin Synthesis

“…Intriguingly, the physiological requirement of biotin in Francisella (Feng et al., 2014) is much less than that of M. smegmatis (Wei et al., 2018) and Agrobacterium tumefaciens (Feng et al., 2013). That is because a single biotinylated AccB protein is predicted, whereas multiple biotinylated enzymes of M. smegmatis (Wei et al., 2018) and A. tumefaciens (Feng et al., 2013) are experimentally verified. Clearly, non-homologous isoenzymes of BioJ gatekeeper of biotin synthesis are distributed within a family of diversified pathogens (Figure 1), which is partially featuring with BioH of Salmonella and Vibrio , BioG of Campylobacter and Neisseria , and BioV of Helicobacter .…”

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation

“…It is unusual, but not without any precedent. In fact, our research group very recently affirmed the presences of such a modification of K47 in the M. smegmatis BioQ that regulates biotin metabolism (Tang et al, 2014; Wei et al, 2018). Given that K134 is conserved in NrtR orthologs of different origins (Figure 7—figure supplement 1A), we hypothesized that it is a common hallmark for NrtR.…”

“…The bacterial species used in this study include E. coli and M. smegmatis (Supplementary file 1). Strains were cultured as described previously (Tang et al, 2014; Wei et al, 2018). pET28a- nrtR and pMV261- nrtR were constructed as follows.…”

“…The recombinant MsNrtR expressed in E. coli and M. smegmatis was separated by SDS-PAGE gel and subjected to peptide mass fingerprinting with Liquid Chromatography (LC)-mass spectrometry (MS) (Wei et al, 2018; Gao et al, 2016a). The resultant polypeptides were separated by the EASY-nLC HPLC system (Thermo Scientific, USA) and detected using a Thermo Fisher LTQ orbitrap elite mass spectrometer (Thermo Scientific, USA).…”

“…The DNA probe containing an MsNrtR-recognizable palindrome (designated nrtR ) was generated by annealing two complementary primers ( nrtR -probe-F and nrtR -probe-R) in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 8.0]) (Gao et al, 2016b). The DNA probe was mixed with purified NrtR protein in EMSA buffer (50 mM Tris-HCl [pH 7.5]; 10 mM MgCl 2 ; 1 mM DDT; 100 mM NaCl) and incubated at room temperature for about 30 min (Wei et al, 2018). The DNA–protein complexes were separated on native 8% native polyacrylamide gels, and the shifted DNA bands were visualized by staining with ethidium bromide (EB) (Gao et al, 2017).…”