Differential expression of the multiple chaperonins of Mycobacterium smegmatis

Rao, Tara; Lund, Peter A.

doi:10.1111/j.1574-6968.2010.02039.x

Cited by 25 publications

(25 citation statements)

References 39 publications

(60 reference statements)

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“…In addition, GroELs generally require a cochaperonin, GroES, to assist in the folding of target proteins. The genome sequence of M. smegmatis strain mc 2 155 has only one copy of the groES gene, Msmeg_1582 (20,27). This groES gene was cloned into MCS-2 of the pCDFDuet-1 vector (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Similar to this, fusion of MimB to an appropriate tag sequence might lead to its high-level soluble expression. Also, the genome sequence of M. smegmatis strain mc 2 155 has two other copies of the groEL gene, Msmeg_0880 and Msmeg_1583, in addition to mimG (Msmeg_1978) (27). These mycobacterial chaperonins might be helpful in the solubilization of MimB.…”

Section: Discussionmentioning

confidence: 99%

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Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Furuya

Hayashi

Kino

2013

Appl Environ Microbiol

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Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Furuya

Hayashi

Kino

2013

Appl Environ Microbiol

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“…These studies have identified two major regulons for heat-shock genes, the HrcA and HspR regulons, which have been demonstrated to control the expression of groE and dnaKJE operons, respectively (Stewart et al 2002). Mtb encodes two copies of groEL genes, groEL1 (cpn60.1) and groEL2 (cpn60.2), and one groES (cpn10) gene, all regulated by HrcA (Rao and Lund 2010). As per the convention, groEL gene that is in operonic arrangement with groES has been termed groEL1, while that that exists separately on the genome has been termed groEL2 (Kong et al 1993;Cole et al 1998).…”

Section: Multiple Groels In Mycobacteriamentioning

confidence: 99%

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

Kumar

Mande

Mahajan

2015

Cell Stress and Chaperones

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Chaperonins are a class of molecular chaperones that assemble into a large double ring architecture with each ring constituting seven to nine subunits and enclosing a cavity for substrate encapsulation. The well-studied Escherichia coli chaperonin GroEL binds non-native substrates and encapsulates them in the cavity thereby sequestering the substrates from unfavorable conditions and allowing the substrates to fold. Using this mechanism, GroEL assists folding of about 10-15 % of cellular proteins. Surprisingly, about 30 % of the bacteria express multiple chaperonin genes. The presence of multiple chaperonins raises questions on whether they increase general chaperoning ability in the cell or have developed specific novel cellular roles. Although the latter view is widely supported, evidence for the former is beginning to appear. Some of these chaperonins can functionally replace GroEL in E. coli and are generally indispensable, while others are ineffective and likewise are dispensable. Additionally, moonlighting functions for several chaperonins have been demonstrated, indicating a functional diversity among the chaperonins. Furthermore, proteomic studies have identified diverse substrate pools for multiple chaperonins. We review the current perception on multiple chaperonins and their physiological and functional specificities.

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“…Mycobacteria are reported to possess multiple copies of GroELs (23,24). Mycobacterium tuberculosis encodes two copies of groEL, of which groEL1 is arranged in an operon with groES and is dispensable whereas groEL2 is located separately on the genome and is indispensable (25,26).…”

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confidence: 99%

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

2016

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Intracellular protein folding is mediated by molecular chaperones, the best studied among which are the chaperonins GroEL and GroES. Conformational changes and allosteric transitions between different metastable states are hallmarks of the chaperonin mechanism. These conformational transitions between three structural domains of GroEL are anchored at two hinges. Although hinges are known to be critical for mediating the communication between different domains of GroEL, the relative importance of hinges on GroEL oligomeric assembly, ATPase activity, conformational changes, and functional activity is not fully characterized. We have exploited the inability of Mycobacterium tuberculosis GroEL2 to functionally complement an Escherichia coli groEL mutant to address the importance of hinge residues in the GroEL mechanism. Various chimeras of M. tuberculosis GroEL2 and E. coli GroEL allowed us to understand the role of hinges and dissect the consequences of oligomerization and substrate binding capability on conformational transitions. The present study explains the concomitant conformational changes observed with GroEL hinge variants and is best supported by the normal mode analysis. IMPORTANCEConformational changes and allosteric transitions are hallmarks of the chaperonin mechanism. We have exploited the inability of M. tuberculosis GroEL2 to functionally complement a strain of E. coli in which groEL expression is repressed to address the importance of hinges. The significance of conservation at the hinge regions stands out as a prominent feature of the GroEL mechanism in binding to GroES and substrate polypeptides. The hinge residues play a significant role in the chaperonin activity in vivo and in vitro. M olecular chaperones are helper proteins, which play essential roles in folding, assembly, and transport of several cellular proteins (1, 2). Chaperonins, a subclass of the molecular chaperones, are homo-or hetero-oligomeric proteins, which carry out the substrate protein folding in a sequestered cavity. One of the best-characterized chaperonins is the 60-kDa chaperonin of Escherichia coli, GroEL (3). Chaperonins are highly conserved proteins and are known to interact with nonnative substrate proteins in an ATP-dependent manner. The E. coli genome possesses a single copy of groEL, arranged in an operonic arrangement with groES and expressed under all growth conditions (4). GroEL forms a cylindrical assembly with two heptameric rings and functions in coordination with the heptameric GroES (5-7). Each subunit of GroEL comprises three domains: (i) an equatorial domain (residues 2 to 133 and 409 to 548), which exhibits ATPase activity and participates in intersubunit and interring interactions; (ii) an apical domain (residues 191 to 374), which binds to substrate polypeptide and cochaperonin GroES; and (iii) an intermediate domain (residues 134 to 190 and 375 to 408), which links apical and equatorial domains in sequence and structure and mediates allosteric communication between the two (8, 9). The two rings...

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Differential expression of the multiple chaperonins of Mycobacterium smegmatis

Cited by 25 publications

References 39 publications

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

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