Structural and Functional Analysis of Rv3214 from Mycobacterium tuberculosis, a Protein with Conflicting Functional Annotations, Leads to Its Characterization as a Phosphatase

ABSTRACT The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4′-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; Rfree = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM α/β structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4′-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

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Implementation of homology based and non-homology based computational methods for the identification and annotation of orphan enzymes: using Mycobacterium tuberculosis H37Rv as a case study

BMC Bioinformatics ◽

10.1186/s12859-020-03794-x ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Swati Sinha ◽

Andrew M. Lynn ◽

Dhwani K. Desai

Keyword(s):

Mycobacterium Tuberculosis ◽

Functional Annotation ◽

Drug Targets ◽

Sequence Similarity ◽

Hole Filling ◽

Mycobacterium Tuberculosis H37rv ◽

Genome Data ◽

Candidate Protein ◽

Computational Procedures

Abstract Background Homology based methods are one of the most important and widely used approaches for functional annotation of high-throughput microbial genome data. A major limitation of these methods is the absence of well-characterized sequences for certain functions. The non-homology methods based on the context and the interactions of a protein are very useful for identifying missing metabolic activities and functional annotation in the absence of significant sequence similarity. In the current work, we employ both homology and context-based methods, incrementally, to identify local holes and chokepoints, whose presence in the Mycobacterium tuberculosis genome is indicated based on its interaction with known proteins in a metabolic network context, but have not been annotated. We have developed two computational procedures using network theory to identify orphan enzymes (‘Hole finding protocol’) coupled with the identification of candidate proteins for the predicted orphan enzyme (‘Hole filling protocol’). We propose an integrated interaction score based on scores from the STRING database to identify candidate protein sequences for the orphan enzymes from M. tuberculosis, as a case study, which are most likely to perform the missing function. Results The application of an automated homology-based enzyme identification protocol, ModEnzA, on M. tuberculosis genome yielded 56 novel enzyme predictions. We further predicted 74 putative local holes, 6 choke points, and 3 high confidence local holes in the genome using ‘Hole finding protocol’. The ‘Hole-filling protocol’ was validated on the E. coli genome using artificial in-silico enzyme knockouts where our method showed 25% increased accuracy, compared to other methods, in assigning the correct sequence for the knocked-out enzyme amongst the top 10 ranks. The method was further validated on 8 additional genomes. Conclusions We have developed methods that can be generalized to augment homology-based annotation to identify missing enzyme coding genes and to predict a candidate protein for them. For pathogens such as M. tuberculosis, this work holds significance in terms of increasing the protein repertoire and thereby, the potential for identifying novel drug targets.

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The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

Infection and Immunity ◽

10.1128/iai.71.7.3794-3801.2003 ◽

2003 ◽

Vol 71 (7) ◽

pp. 3794-3801 ◽

Cited By ~ 49

Author(s):

Tatiana D. Sirakova ◽

Vinod S. Dubey ◽

Hwa-Jung Kim ◽

Michael H. Cynamon ◽

Pappachan E. Kolattukudy

Keyword(s):

Fatty Acids ◽

Mycobacterium Tuberculosis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Immunoblot Analysis ◽

Open Reading Frame ◽

Pcr Analysis ◽

Reading Frame ◽

Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

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pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

Journal of Bacteriology ◽

10.1128/jb.183.2.628-636.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 628-636 ◽

Cited By ~ 31

Author(s):

Sara Lázaro ◽

Francisca Fernández-Piñas ◽

Eduardo Fernández-Valiente ◽

Amaya Blanco-Rivero ◽

Francisco Leganés

Keyword(s):

Sequence Similarity ◽

Shuttle Vector ◽

Molecular Size ◽

Nitrogen Deprivation ◽

Wild Type ◽

Reading Frame ◽

Cyanobacterium Anabaena ◽

In The Wild ◽

Peptidoglycan Layer

ABSTRACT Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequencedAnabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and theEscherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal aspbpB and its product as PBP2. The wild-type form ofpbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N2, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

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Transcriptional Analysis of the Principal Cell Division Gene, ftsZ, of Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.187.7.2540-2550.2005 ◽

2005 ◽

Vol 187 (7) ◽

pp. 2540-2550 ◽

Cited By ~ 12

Author(s):

Sougata Roy ◽

Parthasarathi Ajitkumar

Keyword(s):

Mycobacterium Tuberculosis ◽

Cell Division ◽

Growth Conditions ◽

Principal Cell ◽

Transcriptional Analysis ◽

Upstream Region ◽

Reading Frame ◽

Multiple Promoters ◽

Promoter Fusion

ABSTRACT Multiple promoters drive the expression of the principal cell division gene, ftsZ, in bacterial systems. Primer extension analysis of total RNA from Mycobacterium tuberculosis and a Mycobacterium smegmatis transformant containing 1.117 kb of the upstream region of M. tuberculosis ftsZ and promoter fusion studies identified six ftsZ transcripts and their promoters in the ftsQ open reading frame and ftsQ-ftsZ intergenic region. The presence of multiple promoters reflects the requirement to maintain a high basal level of, or to differentially regulate, FtsZ expression during different growth conditions of the pathogen in vivo.

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Flanking Regulatory Sequences of theTetrahymena R Deletion Element Determine the Boundaries of DNA Rearrangement

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5631 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5631-5641 ◽

Cited By ~ 36

Author(s):

Douglas L. Chalker ◽

Antonietta La Terza ◽

Allison Wilson ◽

Christopher D. Kroenke ◽

Meng-Chao Yao

Keyword(s):

Sequence Similarity ◽

Common Mechanism ◽

Complex Nature ◽

Regulatory Sequence ◽

Regulatory Sequences ◽

Flanking Region ◽

The Many ◽

The Right ◽

Flanking Regions

ABSTRACT In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the leftcis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymenarearrangement.

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Sequence of human carboxypeptidase D reveals it to be a member of the regulatory carboxypeptidase family with three tandem active site domains

Biochemical Journal ◽

10.1042/bj3270081 ◽

1997 ◽

Vol 327 (1) ◽

pp. 81-87 ◽

Cited By ~ 56

Author(s):

Fulong TAN ◽

Michael REHLI ◽

Stefan W. KRAUSE ◽

Randal A. SKIDGEL

Keyword(s):

Active Site ◽

Sequence Similarity ◽

Northern Blotting ◽

Active Site Residues ◽

Virus Binding ◽

Ph Optimum ◽

Hydrophobic Region ◽

Reading Frame ◽

Carboxypeptidase D ◽

Domain 3

We have cloned the cDNA for human carboxypeptidase D (CPD), a new B-type metallocarboxypeptidase that is membrane bound and has an acidic pH optimum. The 5.8 kb of cDNA sequenced contains an open reading frame of 4131 bp encoding 1377 amino acid residues. The sequence is similar (75% identity) to duck gp180, a protein that was isolated, cloned and sequenced as a hepatitis B virus-binding protein but not characterized as a carboxypeptidase. Hydropathic analysis revealed a hydrophobic region at the N-terminus, representing the signal peptide, and one near the C-terminus that probably represents the transmembrane anchor. The most striking feature is the presence of three tandem carboxypeptidase homology domains that have sequence similarity to the regulatory B-type carboxypeptidase family, typified by carboxypeptidases M, E and N. Because of the three repeats, CPD is about three times larger (175–180 kDa) than other members of this family (approx. 50–62 kDa). Domain 2 is most closely related to carboxypeptidases M, E and N (45–48% identity), followed by domain 1 (37–38%) and domain 3 (20–27%). There is much higher sequence identity in regions containing putative active site residues, and all catalytically important residues are strictly conserved in domains 1 and 2. In domain 3, however, only 1 of 8 active site residues is conserved, indicating that this portion might not be catalytically active. Northern blotting of mRNA from human tissues and cells showed high levels of CPD mRNA in placenta, pancreas and Hep G2 hepatoma cells, and smaller amounts in skeletal muscle, heart and HT-29 colon carcinoma and melanoma cell lines.

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Demonstration of In vivo Expression of a Hypothetical Open Reading Frame (ORF-14) Encoded by the RD1 Region of Mycobacterium tuberculosis

Scandinavian Journal of Immunology ◽

10.1111/j.1365-3083.2007.01961.x ◽

2007 ◽

Vol 66 (4) ◽

pp. 422-425 ◽

Cited By ~ 10

Author(s):

H. A. Amoudy ◽

S. Ahmad ◽

J. E. Thole ◽

A. S. Mustafa

Keyword(s):

Mycobacterium Tuberculosis ◽

Open Reading Frame ◽

Reading Frame ◽

In Vivo Expression

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Demonstration of In vivo Expression of a Hypothetical Open Reading Frame (ORF-14) Encoded by the RD1 Region of Mycobacterium tuberculosis

Scandinavian Journal of Immunology ◽

10.1111/j.1365-3083.2007.02023.x ◽

2007 ◽

Vol 66 (5) ◽

pp. 601-601

Keyword(s):

Mycobacterium Tuberculosis ◽

Open Reading Frame ◽

Reading Frame ◽

In Vivo Expression

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Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

Biochemical Journal ◽

10.1042/bj20070770 ◽

2008 ◽

Vol 410 (2) ◽

pp. 291-299 ◽

Cited By ~ 11

Author(s):

Fui-Ching Tan ◽

Qi Cheng ◽

Kaushik Saha ◽

Ilka U. Heinemann ◽

Martina Jahn ◽

...

Keyword(s):

Expression Vector ◽

Fluorescent Protein ◽

Sequence Similarity ◽

Homology Search ◽

Protein Database ◽

Gene Encoding ◽

Reading Frame ◽

Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

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