BCRC Strain Collection Catalog & Shopping Cart

Cambi A, Vincenzetti S, De Sanctis G, Neuhard J, Natalini P, Vita A, ( 2001 )

Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.

Protein engineering 14 (10)
PMID : 11739901 :

Abstract >>

We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.

Keyword	MeSH Terms

Hill JE, Penny SL, Crowell KG, Goh SH, Hemmingsen SM, ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485 : DOI : 10.1101/gr.2649204 PMC : PMC509277

Abstract >>

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.

Keyword	MeSH Terms

Brocca S, Ferrari C, Barbiroli A, Pesce A, Lotti M, Nardini M, ( 2016 )

A bacterial acyl aminoacyl peptidase couples flexibility and stability as a result of cold adaptation.

The FEBS journal 283 (23)
PMID : 27739253 : DOI : 10.1111/febs.13925

Abstract >>

Life in cold environments requires an overall increase in the flexibility of macromolecular and supramolecular structures to allow biological processes to take place at low temperature. Conformational flexibility supports high catalytic rates of enzymes in the cold but in several cases is also a cause of instability. The three-dimensional structure of the psychrophilic acyl aminoacyl peptidase from Sporosarcina psychrophila (SpAAP) reported in this paper highlights adaptive molecular changes resulting in a fine-tuned trade-off between flexibility and stability. In its functional form SpAAP is a dimer, and an increase in flexibility is achieved through loosening of intersubunit hydrophobic interactions. The release of subunits from the quaternary structure is hindered by an 'arm exchange' mechanism, in which a tiny structural element at the N terminus of one subunit inserts into the other subunit. Mutants lacking the 'arm' are monomeric, inactive and highly prone to aggregation. Another feature of SpAAP cold adaptation is the enlargement of the tunnel connecting the exterior of the protein with the active site. Such a wide channel might compensate for the reduced molecular motions occurring in the cold and allow easy and direct access of substrates to the catalytic site, rendering transient movements between domains unnecessary. Thus, cold-adapted SpAAP has developed a molecular strategy unique within this group of proteins: it is able to enhance the flexibility of each functional unit while still preserving sufficient stability. Structural data are available in the Protein Data Bank under the accession number 5L8S.

Keyword	MeSH Terms
acyl aminoacyl peptidase arm exchange cold adaptation α/β hydrolase domain β-propeller domain	Adaptation, Physiological Cold Temperature

Brunialti EA, Gatti-Lafranconi P, Lotti M, ( 2011 )

Promiscuity, stability and cold adaptation of a newly isolated acylaminoacyl peptidase.

Biochimie 93 (9)
PMID : 21635934 : DOI : 10.1016/j.biochi.2011.05.010

Abstract >>

We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low T(m), optimal temperature of 40 �XC, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with k(cat)/K(m) values for ester hydrolysis and acylamino acids cleavage of 1.7 �� 10(4) s(-1) M(-1) and 6.2 �� 10(3) s(-1) M(-1), respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures.

Keyword	MeSH Terms
	Cold Temperature

Tanaka K, Sakai H, Ohta T, Matsuzawa H, ( 1995 )

Molecular cloning of the genes for pyruvate kinase of two bacilli, Bacillus psychrophilus and Bacillus licheniformis, and comparison of the properties of the enzymes produced in Escherichia coli.

Bioscience, biotechnology, and biochemistry 59 (8)
PMID : 7549104 : DOI : 10.1271/bbb.59.1536

Abstract >>

The genes for the pyruvate kinases of a psychrophile, Bacillus psychrophilus, and a mesophile, Bacillus licheniformis, have been cloned in Escherichia coli, and all their nucleotides were sequenced. The two bacterial enzymes each had an extra C-terminal sequence consisting of about 110 amino acid residues, which has been found in the B. stearothermophilus enzyme. Both enzymes were overexpressed in E. coli and the properties of the purified enzymes were compared to those of the B. stearothermophilus enzyme. Both enzymes were less stable than the B. stearothermophilus one. The B. psychrophilus enzyme was more stable than the B. licheniformis one. Similarly to the B. licheniformis and B. stearothermophilus pyruvate kinases, the B. psychrophilus enzyme was activated by AMP or ribose 5-phosphate, and inhibited by ATP or fructose 1,6-bisphosphate. Thus, these enzymes were very similar in the sigmoidal saturation curve for phosphoenolpyruvate and allosteric effectors, but their optimum temperatures and thermostabilities were very different.

Keyword	MeSH Terms