scholarly journals The de novo design of a biocompatible and functional integral membrane protein using minimal sequence complexity

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Christophe J. Lalaurie ◽  
Virginie Dufour ◽  
Anna Meletiou ◽  
Sarah Ratcliffe ◽  
Abigail Harland ◽  
...  
Keyword(s):  
Membrane Protein ◽  
De Novo ◽  
Integral Membrane Protein ◽  
De Novo Design ◽  
Functional Integral ◽  
Sequence Complexity ◽  
Minimal Sequence

scholarly journals Insights into the Relationship between Cobamide Synthase and the Cell Membrane

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Cell Membrane ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
De Novo ◽  
Integral Membrane Protein ◽  
Phospholipid Bilayer ◽  
Multienzyme Complex ◽  
Coenzyme B ◽  
Lower Ligand

ABSTRACT Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The “late steps” of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5′ phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis. IMPORTANCE Salmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

scholarly journals Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jacob E. Choby ◽  
Caroline M. Grunenwald ◽  
Arianna I. Celis ◽  
Svetlana Y. Gerdes ◽  
Jennifer L. DuBois ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Membrane Protein ◽  
De Novo ◽  
Common Factor ◽  
Integral Membrane Protein ◽  
Heme Biosynthesis ◽  
Bacterial Physiology ◽  
Heme Synthesis ◽  
Regulatory Processes ◽  
High Concentrations

ABSTRACTStaphylococcus aureusis responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesizedde novofrom common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme homeostasis. In this work, we describe a posttranscriptional regulatory strategy inS. aureusheme biosynthesis. The first committed enzyme in theS. aureusheme biosynthetic pathway, glutamyl-tRNA reductase (GtrR), is regulated by heme abundance and the integral membrane protein HemX. GtrR abundance increases dramatically in response to heme deficiency, suggesting a mechanism by whichS. aureusresponds to the need to increase heme synthesis. Additionally, HemX is required to maintain low levels of GtrR in heme-proficient cells, and inactivation ofhemXleads to increased heme synthesis. Excess heme synthesis in a ΔhemXmutant activates the staphylococcal heme stress response, suggesting that regulation of heme synthesis is critical to reduce self-imposed heme toxicity. Analysis of diverse organisms indicates that HemX is widely conserved among heme-synthesizing bacteria, suggesting that HemX is a common factor involved in the regulation of GtrR abundance. Together, this work demonstrates thatS. aureusregulates heme synthesis by modulating GtrR abundance in response to heme deficiency and through the activity of the broadly conserved HemX.IMPORTANCEStaphylococcus aureusis a leading cause of skin and soft tissue infections, endocarditis, bacteremia, and osteomyelitis, making it a critical health care concern. Development of new antimicrobials againstS. aureusrequires knowledge of the physiology that supports this organism’s pathogenesis. One component of staphylococcal physiology that contributes to growth and virulence is heme. Heme is a widely utilized cofactor that enables diverse chemical reactions across many enzyme families.S. aureusrelies on many critical heme-dependent proteins and is sensitive to excess heme toxicity, suggestingS. aureusmust maintain proper intracellular heme homeostasis. BecauseS. aureusprovides heme for heme-dependent enzymes via synthesis from common precursors, we hypothesized that regulation of heme synthesis is one mechanism to maintain heme homeostasis. In this study, we identify thatS. aureusposttranscriptionally regulates heme synthesis by restraining abundance of the first heme biosynthetic enzyme, GtrR, via heme and the broadly conserved membrane protein HemX.

scholarly journals SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast

1993 ◽  
Vol 122 (1) ◽  
pp. 79-94 ◽  
Author(s):  
EA Whitters ◽  
AE Cleves ◽  
TP McGee ◽  
HB Skinner ◽  
VA Bankaitis
Keyword(s):  
Membrane Protein ◽  
Actin Cytoskeleton ◽  
Protein Function ◽  
De Novo ◽  
Integral Membrane Protein ◽  
Secretory Function ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Golgi Secretory Function

Mutations in the SAC1 gene exhibit allele-specific genetic interactions with yeast actin structural gene defects and effect a bypass of the cellular requirement for the yeast phosphatidylinositol/phosphatidylcholine transfer protein (SEC14p), a protein whose function is essential for sustained Golgi secretory function. We report that SAC1p is an integral membrane protein that localizes to the yeast Golgi complex and to the yeast ER, but does not exhibit a detectable association with the bulk of the yeast F-actin cytoskeleton. The data also indicate that the profound in vivo effects on Golgi secretory function and the organization of the actin cytoskeleton observed in sac1 mutants result from loss of SAC1p function. This cosuppression of actin and SEC14p defects is a unique feature of sac1 alleles as mutations in other SAC genes that result in a suppression of actin defects do not result in phenotypic suppression of SEC14p defects. Finally, we report that sac1 mutants also exhibit a specific inositol auxotrophy that is not exhibited by the other sac mutant strains. This sac1-associated inositol auxotrophy is not manifested by measurable defects in de novo inositol biosynthesis, nor is it the result of some obvious defect in the ability of sac1 mutants to utilize inositol for phosphatidylinositol biosynthesis. Thus, sac1 mutants represent a novel class of inositol auxotroph in that these mutants appear to require elevated levels of inositol for growth. On the basis of the collective data, we suggest that SAC1p dysfunction exerts its pleiotropic effects on yeast Golgi function, the organization of the actin cytoskeleton, and the cellular requirement for inositol, through altered metabolism of inositol glycerophospholipids.

scholarly journals Protein storage vacuoles form de novo during pea cotyledon development

1995 ◽  
Vol 108 (1) ◽  
pp. 299-310 ◽  
Author(s):  
B. Hoh ◽  
G. Hinz ◽  
B.K. Jeong ◽  
D.G. Robinson
Keyword(s):  
Membrane Protein ◽  
De Novo ◽  
Storage Proteins ◽  
Protein Body ◽  
Membrane System ◽  
Intermediate Stage ◽  
Integral Membrane Protein ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Protein Storage Vacuole

We have investigated the formation of protein storage vacuoles in peas (Pisum sativum L.) in order to determine whether this organelle arises de novo during cotyledon development. A comparison of different stages in cotyledon development indicates that soluble protease activities decline and the amounts of storage proteins and the integral membrane protein of the protein body, alpha-TIP, increase during seed maturation. On linear sucrose density gradients we have been able to distinguish between two separate vesicle populations: one enriched in alpha-TIP, and one in TIP-Ma 27, a membrane protein characteristic of vegetative vacuoles. Both vesicle populations possess, however, PPase and V-ATPase activities. Conventionally fixed cotyledonary tissue at an intermediate stage in cotyledon development reveals the presence of a complex tubular-cisternal membrane system that seems to surround the pre-existing vacuoles. The latter gradually become compressed as a result of dilation of the former membrane system. This was confirmed immunocytochemically with the TIP-Ma 27 antiserum. Deposits of the storage proteins vicilin and legumin in the lumen, and the presence of alpha-TIP in the membranes of the expanding membrane system provide evidence of its identity as a precursor to the protein storage vacuole.

scholarly journals The CbiB Protein of Salmonella enterica Is an Integral Membrane Protein Involved in the Last Step of the De Novo Corrin Ring Biosynthetic Pathway

2007 ◽  
Vol 189 (21) ◽  
pp. 7697-7708 ◽  
Author(s):  
Carmen L. Zayas ◽  
Kathy Claas ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Membrane Protein ◽  
Salmonella Enterica ◽  
De Novo ◽  
Integral Membrane Protein ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Serovar Typhimurium ◽  
Ethanolamine Phosphate ◽  
Coenzyme B

ABSTRACT We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamide-phosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B12) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but l-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

Sign in / Sign up

Export Citation Format

Share Document