Abstract
BackgroundThe eighth step of l-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in l-histidine biosynthesis.ResultsDeletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete l-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases.Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases.ConclusionThe kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes.
Highlights
The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase)
Genetic study on hisN and its four paralogs During our previous investigation of different L-histidine gene deletion mutants of C. glutamicum [5], we observed that deletion of hisN, encoding the inositol monophosphatase (IMPase)-like HolPase, does not result in L-histidine auxotrophy, but only in a pronounced bradytrophy of the mutant
We started a closer investigation of the 8th step of L-histidine biosynthesis in C. glutamicum in general and the ΔhisN mutant in particular
Summary
The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). The third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. There exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in L-histidine biosynthesis. All organisms known to synthesize L-histidine, including archaea, bacteria, yeast, and plants, use the same pathway for the biosynthesis. C. glutamicum possesses a HolPase belonging to the family of inositol monophosphatase (IMPase)-like proteins, a subgroup of the FIG (FBPase/IMPase/GlpX-like domain) superfamily encoded by hisN [7, 8]. IMPase-like HolPases are a characteristic of the Actinobacteria and genera possessing a HisN homolog can be found in almost all taxonomical orders of this bacterial class [5]. The HolPase activity of the HisN homolog in Mycobacterium tuberculosis (gene Rv3137) is supported at least indirectly, since it is not possible to delete this gene if a L-histidine free medium is used during the required selection steps [11]
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