Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Chaturvedi, Sarita; Bhakuni, Vinod

doi:10.1074/jbc.m306192200

Cited by 33 publications

(25 citation statements)

References 28 publications

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“…These include three predicted surface proteins of unknown function: a member of the PPE family (PPE10), a lipoprotein (LppN), and a conserved hypothetical protein (Rv3707c). The list includes two secreted enzymes: cut2, encoding a member of a family of serine esterases with significant similarity to fungal cutinases, and glyA1, a serine hydroxymethyltransferase that is found in the culture filtrate of M. tuberculosis [43]. Insertion in kefB provides the most obvious connection between gene function and altered phagosomal pH.…”

Section: Resultsmentioning

confidence: 99%

Mycobacterial Mutants with Defective Control of Phagosomal Acidification

et al. 2005

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The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

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Section: Resultsmentioning

confidence: 99%

Mycobacterial Mutants with Defective Control of Phagosomal Acidification

et al. 2005

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“…The gcvT element often occurs adjacent to genes for putative Na ϩ ͞ alanine symporters, and in one instance it is located upstream of ␥-aminobutyrate permease. The element also occurs upstream of a redundant serine hydroxymethyltransferase gene (28) and L-serine deaminase gene in Mycobacterium tuberculosis, and a glycine͞D-amino acid oxidase gene in Mesorhizobium loti. Both of these latter systems are used to convert glycine into pyruvate.…”

Section: Resultsmentioning

confidence: 99%

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control

Barrick

Corbino

Winkler

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

452

500

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The expression of certain genes involved in fundamental metabolism is regulated by metabolite-binding ''riboswitch'' elements embedded within their corresponding mRNAs. We have identified at least six additional elements within the Bacillus subtilis genome that exhibit characteristics of riboswitch function (glmS, gcvT, ydaO͞yuaA, ykkC͞yxkD, ykoK, and yybP͞ykoY). These motifs exhibit extensive sequence and secondary-structure conservation among many bacterial species and occur upstream of related genes. The element located upstream of the glmS gene in Grampositive organisms functions as a metabolite-dependent ribozyme that responds to glucosamine-6-phosphate. Other motifs form complex folded structures when transcribed as RNA molecules and carry intrinsic terminator structures. These findings indicate that riboswitches serve as a major genetic regulatory mechanism for the control of metabolic genes in many microbial species. R iboswitches are highly structured domains within mRNAs that precisely sense metabolites and control gene expression (1). These RNA elements are capable of binding to a variety of target compounds and subsequently modulating transcription and translation with performance characteristics that are similar to those of protein genetic factors. Typically, each riboswitch is composed of a conserved metabolite-binding domain (aptamer) located upstream of a variable sequence region (expression platform) that dictates the level of gene expression. Allosteric changes brought about by metabolite binding to the aptamer are harnessed by the expression platform to modulate the expression of the adjacent gene or operon. Riboswitches are versatile genetic control elements. In some instances, both transcription and translation control are used by the same aptamer class in the same prokaryotic organism (e.g., see ref.2). Evidence also shows that riboswitches can use mRNA-processing events to modulate gene expression (3, 4).The various metabolites that are detected by known riboswitches are of fundamental importance to living systems (5). On this basis, we have speculated that modern riboswitches might be the remaining representatives of an ancient metabolitemonitoring system that was present in the RNA World (5-9). The wide distribution of some riboswitch classes among microbes (e.g., see refs. 5 and 9-14) and the presence of metabolitebinding RNA domains in eukaryotes (4) support this hypothesis. Each of the seven classes of riboswitches reported (1, 5) was examined for metabolite-binding function because published genetic evidence showed that these elements were important for genetic control. Because the regulation of many metabolism genes has not been characterized in detail, it is possible that numerous other metabolite-binding RNA motifs exist in nature.The riboswitches known to be present in prokaryotes are typically located in noncoding or intergenic regions (IGRs). Therefore, the examination of unusually long IGRs for indications of conserved sequence and secondary-structure elements should yield new...

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“…The cross-linking of protein samples was carried out with 1% glutaraldehyde as described earlier (24). The molecular mass of the cross-linked products were determined by 10% SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Unusual Structural Features of the Bacteriophage-associated Hyaluronate Lyase (hylp2)

Mishra¹,

Akhtar

Bhakuni

2006

Journal of Biological Chemistry

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Hyaluronate lyases are a class of endoglycosaminidase enzymes, which are of considerable complexity and heterogeneity. Their primary function is to degrade hyaluronan and certain other glycosaminoglycans and facilitate the spread of disease. Among hyaluronate lyases, the bacteriophage-associated enzymes are unique as they have the lowest molecular mass, very low amino acid sequence homology with bacterial hyaluronate lyases, and exhibit absolute specificity for one type of glycosaminoglycan, i.e. hyaluronan. Despite such unique characteristics significant details on structural features of these lyases are not available. The Streptococcus pyogenes bacteriophage 10403 contains a gene, hylP2, which encodes for hyaluronate lyase (HylP2) in this organism. HylP2 was cloned, overexpressed, and purified to homogeneity. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa, under physiological conditions. Limited proteolysis and guanidine hydrochloride denaturation studies demonstrated that the N-terminal region of the protein is flexible, whereas the C-terminal portion has a compact conformation. The enzyme shows sequential unfolding, with the N-terminal unfolding first followed by the simultaneous unfolding and dissociation of the stabilized trimeric C-terminal domain. We isolated a functionally active C-terminal fragment (Ser 128 -Lys 337 ) of the protein that was stabilized in a trimeric configuration. Comparative functional studies with full-length protein, N:C complex, and isolated C-terminal domain demonstrated that the active site of HylP2 is present in the C-terminal portion of the enzyme, and the N-terminal portion modulates the substrate specificity and enzymatic activity of the C-terminal domain.

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Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

Cited by 33 publications

References 28 publications

Mycobacterial Mutants with Defective Control of Phagosomal Acidification

Mycobacterial Mutants with Defective Control of Phagosomal Acidification

New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control

Unusual Structural Features of the Bacteriophage-associated Hyaluronate Lyase (hylp2)

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