Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond

Abstract

Background: The 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. Results: Deletion 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. Conclusion: The 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.

Construction of a complete set of Neisseria meningitidis mutants and its use for the phenotypic profiling of this human pathogen

“…6a). The meningococcal genome does not have homologues of known histidinol-phosphatases 34 , a feature it shares with many other bacterial species according to MetaCyc 25 . First, we excluded the possibility that the meningococcus might be auxotrophic for histidine by showing that 8013 can grow on M9 minimal medium without added histidine (Fig.…”

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

“…Most of the genes and their genomic organization are conserved between phylogenetically diverse species and the corresponding enzymes possess homologous folds (Del Duca et al, 2020 ; Fani et al, 2005 ; Winkler & Ramos‐Montañez, 2009 ). However, there is one exception, namely the HolPase (Brilli & Fani, 2004 ; Kulis‐Horn et al, 2017 ). In E. coli and related γ‐Proteobacteria, the HolPase function is catalyzed by the N‐terminal part of the bi‐functional HisB enzyme and was hence termed HisB‐N, while the C‐terminal part harbors the imidazole glycerol phosphate dehydratase (IGPDH) (Brady & Houston, 1973 ; Brilli & Fani, 2004 ; Chiariotti et al, 1986 ; Chumley & Roth, 1981 ; Fani et al, 2005 ).…”

“…In contrast, the monofunctional HolPase from Lactococcus lactis exhibits a (βα) 7 ‐barrel fold and belongs to the polymerase and histidinol phosphatase (PHP) superfamily (Ghodge et al, 2013 ), and the monofunctional HolPases from Mycobacterium tuberculosis and Medicago truncatula show the fold of a αβαβα‐sandwich and belong to the inositol monophosphatase (IMP) superfamily (Figure S2 ) (Jha et al, 2018 ; Ruszkowski & Dauter, 2016 ). The differences between HolPases also extend to the genomic organization of their respective genes: The gene that encodes ec HisB‐N is part of the his operon, whereas genes of HolPases from the PHP and IMP superfamily are typically located outside of the his operon (Brilli & Fani, 2004 ; Fani et al, 2005 ; Ghodge et al, 2013 ; Kulis‐Horn et al, 2017 ; Mormann et al, 2006 ; Ruszkowski & Dauter, 2016 ). Taken together, these observations indicate that HisB‐N together with other HolPases are evolutionary young compared with the rest of the histidine biosynthetic enzymes.…”

Construction of a complete set ofNeisseria meningitidisdefined mutants – the NeMeSys 2.0 collection – and its use for the phenotypic profiling of the genome of an important human pathogen

“…7a). The meningococcal genome does not have homologs of known histidinol-phosphatases 34 , a feature it shares with many other bacterial species according to MetaCyc . First, we excluded the possibility that the meningococcus might be auxotrophic for histidine by showing that can grow on M9 minimal medium without added histidine (Fig.…”