Structural studies of a cold-adapted dimeric β-D-galactosidase fromParacoccussp. 32d

2016 ◽  
Vol 72 (9) ◽  
pp. 1049-1061 ◽  
Author(s):  
Maria Rutkiewicz-Krotewicz ◽  
Agnieszka J. Pietrzyk-Brzezinska ◽  
Bartosz Sekula ◽  
Hubert Cieśliński ◽  
Anna Wierzbicka-Woś ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Domain Architecture ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
Amino Acid Residues ◽  
Dimer Interface ◽  
Cold Adapted ◽  
Domain 3 ◽  
Hydrolysis Of

The crystal structure of a novel dimeric β-D-galactosidase fromParacoccussp. 32d (ParβDG) was solved in space groupP212121at a resolution of 2.4 Å by molecular replacement with multiple models using theBALBESsoftware. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases fromEscherichia coliandArthrobactersp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG–Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 inParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

A Cold-Adapted Lipase of an Alaskan Psychrotroph,Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

1998 ◽  
Vol 64 (2) ◽  
pp. 486-491 ◽  
Author(s):  
Dong-Won Choo ◽  
Tatsuo Kurihara ◽  
Takeshi Suzuki ◽  
Kenji Soda ◽  
Nobuyoshi Esaki
Keyword(s):  
Amino Acid ◽  
Enzyme Purification ◽  
Hormone Sensitive Lipase ◽  
Amino Acid Residues ◽  
Lipase Gene ◽  
Positional Specificity ◽  
Cold Adapted ◽  
Nitrophenyl Phosphate ◽  
Hormone Sensitive ◽  
Hydrolysis Of

ABSTRACT A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from AntarcticMoraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinantEscherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis ofp-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

scholarly journals Identification and Analysis of Novel Amino-Acid Sequence Repeats inBacillus anthracisstr.AmesProteome Using Computational Tools

2007 ◽  
Vol 2007 ◽  
pp. 1-23 ◽  
Author(s):  
G. R. Hemalatha ◽  
D. Satyanarayana Rao ◽  
L. Guruprasad
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Protein Sequence ◽  
Amino Acid Residues ◽  
Sequence Motifs ◽  
Computational Tools ◽  
Conserved Sequence ◽  
Conserved Sequence Motifs ◽  
Multiple Copies ◽  
Domain 3

We have identified four repeats and ten domains that are novel in proteins encoded by theBacillus anthracisstr.Amesproteome using automated in silico methods. A “repeat” corresponds to a region comprising less than 55-amino-acid residues that occur more than once in the protein sequence and sometimes present in tandem. A “domain” corresponds to a conserved region with greater than 55-amino-acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 57-amino-acid-residue PxV domain, (2) 122-amino-acid-residue FxF domain, (3) 111-amino-acid-residue YEFF domain, (4) 109-amino-acid-residue IMxxH domain, (5) 103-amino-acid-residue VxxT domain, (6) 84-amino-acid-residue ExW domain, (7) 104-amino-acid-residue NTGFIG domain, (8) 36-amino-acid-residue NxGK repeat, (9) 95-amino-acid-residue VYV domain, (10) 75-amino-acid-residue KEWE domain, (11) 59-amino-acid-residue AFL domain, (12) 53-amino-acid-residue RIDVK repeat, (13) (a) 41-amino-acid-residue AGQF repeat and (b) 42-amino-acid-residue GSAL repeat. A repeat or domain type is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.

scholarly journals Crystal structure of enolase fromDrosophila melanogaster

2017 ◽  
Vol 73 (4) ◽  
pp. 228-234 ◽  
Author(s):  
Congcong Sun ◽  
Baokui Xu ◽  
Xueyan Liu ◽  
Zhen Zhang ◽  
Zhongliang Su
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
Structural Basis ◽  
Molecular Replacement ◽  
Biological Processes ◽  
Conserved Residues ◽  
Dimer Interface ◽  
Open Conformation ◽  
Evolutionary Studies ◽  
Important Enzyme

Enolase is an important enzyme in glycolysis and various biological processes. Its dysfunction is closely associated with diseases. Here, the enolase fromDrosophila melanogaster(DmENO) was purified and crystallized. A crystal of DmENO diffracted to 2.0 Å resolution and belonged to space groupR32. The structure was solved by molecular replacement. Like most enolases, DmENO forms a homodimer with conserved residues in the dimer interface. DmENO possesses an open conformation in this structure and contains conserved elements for catalytic activity. This work provides a structural basis for further functional and evolutionary studies of enolase.

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

scholarly journals Structure–function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD

2001 ◽  
Vol 355 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Chang Hoon LEE ◽  
Patrick Y. UM ◽  
Myung Hee PARK
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Binding Sites ◽  
Binding Pocket ◽  
Complete Loss ◽  
Amino Acid Residues ◽  
Deoxyhypusine Synthase ◽  
Positive Identification ◽  
Insight Into

Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305 → Ala showed partial binding and activity. His-288 → Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137 → Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342 → Ala, Asp-313 → Ala and Asp-238 → Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction.

scholarly journals Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

2003 ◽  
Vol 69 (4) ◽  
pp. 2349-2355 ◽  
Author(s):  
Yuji Nagata ◽  
Zbyněk Prokop ◽  
Soňa Marvanová ◽  
Jana Sýkorová ◽  
Marta Monincová ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Active Site ◽  
Homology Model ◽  
Protein Family ◽  
Sphingomonas Paucimobilis ◽  
Amino Acid Residues ◽  
Haloalkane Dehalogenase

ABSTRACT The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.

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