Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi Structural characterization of ribT from Bacillus subtilis reveals it as a GCN5- related N-acetyltransferase Ritika Srivastava, Amanpreet Kaur, Charu Sharma, Subramanian Karthikeyan ⁎ CSIR-Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh 160 036, India ARTICLE INFO Keywords: Riboflavin Crystal structure CoA, GNAT Acetylation ABSTRACT In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5- related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-con- served amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis. 1. Introduction Riboflavin which is also known as vitamin B 2 is an essential com- ponent for all kingdoms of life as it serves as an ultimate precursor for the biosynthesis of two universal and indispensable cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). FMN and FAD play an important role in a variety of redox reactions such as electron transport, photosynthesis, fatty acid oxidation and vitamin metabolism (Mansoorabadi et al., 2007). Interestingly, these coenzymes have also been found to participate in a variety of non-redox reactions like light sensing (Mattevi, 2006), DNA repair (Essen and Klar, 2006), bioluminescence, etc. apart from the conventional redox reactions that they carry out. Many microorganisms and plants can synthesize ribo- flavin de novo, but higher organisms lack this ability, and they need to obtain riboflavin from various dietary sources (Burgess et al., 2006). Owing to its multitudinous functions, riboflavin deficiency is associated with several health related risks. Therefore, riboflavin is produced in thousands of tons every year either chemically or biotechnologically to meet the nutritional requirements and is used in the fortification of food or in medicine or as food colorant (Schwechheimer et al., 2016). Ri- boflavin biosynthesis is best studied in Gram positive bacterium B. subtilis (Perkins and Pero, 2002) which has also been extensively used for the production of riboflavin (Abd-Alla et al., 2016; Stahmann et al., 2000). The riboflavin biosynthesis genes of B. subtilis are clustered in an operon consisting of five non overlapping genes (rib D/GEAHT) (Mironov et al., 1994). These genes encode for bifunctional pyrimidine deaminase/reductase (ribD/G), riboflavin synthase (ribE), bifunctional GTP cyclohydrolase II/ 3, 4-dihydroxy-2-butanone-4-phosphate syn- thase (ribA) and lumazine synthase (ribH). These four enzymes along with an unknown phosphatase collectively catalyse the conversion of GTP and ribulose-5-phosphate to riboflavin (Bacher et al., 2000). Many biochemical and structural studies have been reported for the proteins encoded by these genes in B. subtilis (Chen et al., 2009; Richter et al., 1997) except for bribT. The bribT gene occupies the distal terminal of rib operon which encodes for 124 amino acid protein and predicted to have N-acetyltransferase fold (Yakimov et al., 2014). N-acetyl- transferase catalyses the transfer of acetyl group from AcCoA to a wide range of substrates ranging from small molecules to proteins (Dyda et al., 2000; Vetting et al., 2005). Although a well-known process in eukaryotes, the protein acetylation in bacteria has been recently dis- covered and is known to play variety of roles in various cell physiolo- gies like cell differentiation, cell survival, metabolism, stress response https://doi.org/10.1016/j.jsb.2017.12.006 Received 24 August 2017; Received in revised form 4 December 2017; Accepted 11 December 2017 ⁎ Corresponding author. E-mail address: skarthik@imtech.res.in (S. Karthikeyan). Journal of Structural Biology xxx (xxxx) xxx–xxx 1047-8477/ © 2017 Elsevier Inc. All rights reserved. Please cite this article as: Srivastava, R., Journal of Structural Biology (2017), https://doi.org/10.1016/j.jsb.2017.12.006