Innovative Hybrid-Alignment Annotation Method for Bioinformatics Identification and Functional Verification of a Novel Nitric Oxide Synthase in Trichomonas vaginalis

SIMPLE SUMMARY: Both the annotation and identification of genes in pathogenic parasites remain challenging. As a survival factor, nitric oxide (NO) has been proven to be synthesized in Trichomonas vaginalis (TV). However, nitric oxide synthase (NOS) has not yet been annotated in the TV genome. By aligning whole coding sequences of TV against a thousand sequences of known proteins from other organisms via the Smith–Waterman and Needleman–Wunsch algorithms, we developed a witness-to-suspect strategy to identify incorrectly annotated genes in TV. A novel NOS of TV (TV NOS) with a high witness-to-suspect ratio, which was originally annotated as a hydrogenase in the NCBI database, was successfully identified. We then performed in silico modeling of the protein structure and the molecular docking of all cofactors (NADPH, tetrahydrobiopterin (BH4), heme and flavin adenine dinucleotide (FAD)), cloned the gene, expressed and purified the protein, and ultimately performed mass spectrometry analysis and enzymatic activity assays. We clearly showed that although the predicted structure of TV NOS is not similar to that of NOS proteins of other species, all cofactor-binding motifs can interact with their ligands with high affinities. Most importantly, the purified protein is a functional NOS, as it has a high enzymatic activity for generating NO in vitro. This study provides an innovative approach to identify incorrectly annotated genes. ABSTRACT: Both the annotation and identification of genes in pathogenic parasites are still challenging. Although, as a survival factor, nitric oxide (NO) has been proven to be synthesized in Trichomonas vaginalis (TV), nitric oxide synthase (NOS) has not yet been annotated in the TV genome. We developed a witness-to-suspect strategy to identify incorrectly annotated genes in TV via the Smith–Waterman and Needleman–Wunsch algorithms through in-depth and repeated alignment of whole coding sequences of TV against thousands of sequences of known proteins from other organisms. A novel NOS of TV (TV NOS), which was annotated as hydrogenase in the NCBI database, was successfully identified; this TV NOS had a high witness-to-suspect ratio and contained all the NOS cofactor-binding motifs (NADPH, tetrahydrobiopterin (BH4), heme and flavin adenine dinucleotide (FAD) motifs). To confirm this identification, we performed in silico modeling of the protein structure and cofactor docking, cloned the gene, expressed and purified the protein, performed mass spectrometry analysis, and ultimately performed an assay to measure enzymatic activity. Our data showed that although the predicted structure of the TV NOS protein was not similar to the structure of NOSs of other species, all cofactor-binding motifs could interact with their ligands with high affinities. We clearly showed that the purified protein had high enzymatic activity for generating NO in vitro. This study provides an innovative approach to identify incorrectly annotated genes in TV and highlights a novel NOS that might serve as a virulence factor of TV.