Multiple Proline Substitutions Cumulatively Thermostabilize Bacillus Cereus ATCC7064 Oligo‐1,6‐Glucosidase

Background: Molecular features underlying enzyme function in extreme environments are poorly understood. Results: Identification of the basis for thermostability, halophilicity, and detoxification activity in a mercuric reductase from hot deep-sea brine. Conclusion: A small number of structural modifications accounts for the enzyme's robustness. Significance: This work defines novel adaptations that enable enzymes to cope with multiple abiotic stressors simultaneously.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

Sayed

Ghazy

Ferreira

et al. 2014

“…2). The entropy effect of proline contributes positively in protein stability; thus, mutation at a proline residue might introduce an increased flexibility (50,51). We hypothesize that the openclose conformational switch is essential for the catalytic activity of adenylyl cyclase and that the increased flexibility of the protein by the proline to serine mutation might facilitate such a switch (1,13).…”

Section: Discussionmentioning

confidence: 99%

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Yan

Huang

Shaw

et al. 1997

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C 2 domain is catalytically active and the C 1 domain is not; the role of the C 1 domain is to promote the catalytic activity of the C 2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C 2 domains, but not among the C 1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30 -100-fold reduction in K cat of adenylyl cyclase. However, the same mutations do not affect the K m for ATP, the half-maximal concentration (EC 50 ) for the C 2 domain of type II adenylyl cyclase to associate with the C 1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC 50 for forskolin to maximally activate enzyme activity with or without G s␣ . This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C 1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in K cat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases 1 and guanylyl cyclases (1).All nine isoforms have a similar structure, including two intensely hydrophobic domains (M 1 and M 2 ) and two 40-kDa cytoplasmic domains (C 1 and C 2 ). The two cytoplasmic domains contain sequences (C 1a and C 2a ) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein 2 ␣ subunit, designated as G s␣ , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein ␤␥ subunits, the ␣ subunit of G i , G o , and G z , Ca 2ϩ ion, Ca 2ϩ -calmodulin, Ca 2ϩ -calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. ␤␥) can be either stimulatory or inhibitory...

“…Strategically placed prolines can increase protein thermostability, and isomerization of proline residues can be the ratelimiting step in protein folding (38,39). Evidence (40,41) suggests that inductive effects of electronegative substituents on the proline ring affect the rate and equilibrium position of cis-trans isomerization and, consequently, triple helix stability.…”

Section: Trans-4-hydroxyproline Incorporation In Bacteriamentioning

confidence: 99%

Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Buechter¹,

Paolella²,

Leslie³

et al. 2003

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies.Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitro acylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in sitespecific incorporation (1) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency. In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2-4), but experimental hurdles inherent to this approach have not yet been fully overcome.In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5) and tryptophan (6). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type syntheta...