Sequence Determinants of Substrate Ambiguity in a HAD Phosphosugar Phosphatase of Arabidopsis Thaliana

The Arabidopsis thaliana broad-range sugar phosphate phosphatase AtSgpp (NP_565895.1, locus AT2G38740) and the specific DL-glycerol-3-phosphatase AtGpp (NP_568858.1, locus AT5G57440) are members of the wide family of magnesium-dependent acid phosphatases subfamily I with the C1-type cap domain haloacid dehalogenase-like hydrolase proteins (HAD). Although both AtSgpp and AtGpp have a superimporsable α/β Rossmann core active site, they differ with respect to the loop-5 of the cap domain, accounting for the differences in substrate specificity. Recent functional studies have demonstrated the essential but not sufficient role of the signature sequence within the motif-5 in substrate discrimination. To better understand the mechanism underlying the control of specificity, we explored additional sequence determinants underpinning the divergent evolutionary selection exerted on the substrate affinity of both enzymes. The most evident difference was found in the loop preceding the loop-5 of the cap domain, which is extended in three additional residues in AtSgpp. To determine if the shortening of this loop would constrain the substrate ambiguity of AtSgpp, we deleted these three aminoacids. The kinetic analyses of the resulting mutant protein AtSgpp3Δ (ΔF53, ΔN54, ΔN55) indicate that promiscuity is compromised. AtSgpp3Δ displays highest level of discrimination for D-ribose-5-phosphate compared to the rest of phosphate ester metabolites tested, which may suggest a proper orientation of D-ribose-5-phosphate in the AtSgpp3Δ active site.

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Structural insights into the mechanism defining substrate affinity in Arabidopsis thaliana dUTPase: the role of tryptophan 93 in ligand orientation

BMC Research Notes ◽

10.1186/s13104-015-1760-1 ◽

2015 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Noriko Inoguchi ◽

Kittichai Chaiseeda ◽

Mamoru Yamanishi ◽

Moon Ki Kim ◽

Yunho Jang ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Substrate Affinity ◽

Ligand Orientation ◽

Structural Insights

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Spectroscopic and Density Functional Studies of the Red Copper Site in Nitrosocyanin: Role of the Protein in Determining Active Site Geometric and Electronic Structure

Journal of the American Chemical Society ◽

10.1021/ja044412+ ◽

2005 ◽

Vol 127 (10) ◽

pp. 3531-3544 ◽

Cited By ~ 65

Author(s):

Lipika Basumallick ◽

Ritimukta Sarangi ◽

Serena DeBeer George ◽

Brad Elmore ◽

Alan B. Hooper ◽

...

Keyword(s):

Electronic Structure ◽

Active Site ◽

Density Functional ◽

Functional Studies ◽

Copper Site

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The Role of the Active Site Cysteine in Catalysis by Type 1 Iodothyronine Deiodinase*

Endocrinology ◽

10.1210/endo.138.12.5623 ◽

1997 ◽

Vol 138 (12) ◽

pp. 5452-5458 ◽

Cited By ~ 11

Author(s):

Ben C. Sun ◽

John W. Harney ◽

Marla J. Berry ◽

P. Reed Larsen

Keyword(s):

Active Site ◽

Outer Ring ◽

Substrate Affinity ◽

Maximal Velocity ◽

Wild Type ◽

Iodothyronine Deiodinase ◽

Active Site Cysteine ◽

Enzymatic Function

Abstract Type 1 iodothyronine deiodinase (deiodinase 1) is a selenoenzyme that converts the prohormone T4 to the active thyroid hormone T3 by outer ring deiodination or to the inactive metabolite rT3 by inner ring deiodination. Although selenocysteine has been demonstrated to be essential for the biochemical profile of deiodinase 1, the role of a highly conserved, active site cysteine (C124 in rat deiodinase 1) has not been defined. The present studies examined the effects of a Cys124Ala mutation on rat deiodinase 1 enzymatic function and substrate affinity. At a constant 10-mm concentration of dithiothreitol (DTT), the C124A mutant demonstrated a 2-fold lower apparent maximal velocity (Vmax) and Km for rT3 (KmrT3) than the wild type for outer ring deiodination, whereas the Vmax/Km ratio was unchanged. Similarly, the apparent Vmax and KmT3 sulfate for inner ring deiodination were 2-fold lower in the C124A mutant relative to those in the wild type, with no change in the Vmax/Km ratio. The C124A mutant exhibited ping-pong kinetics in the presence of DTT, and substitution of the active site cysteine increased the KmDTT by 14-fold relative to that of the wild-type enzyme, with no significant effects on KmrT3 or Vmax. The C124A mutant was inhibited by propylthiouracil in an uncompetitive fashion and exhibited a 2-fold increase in Kipropylthiouracil compared with that of the wild type. KmrT3 was also reduced for the C124A mutant when 5 mm reduced glutathione, a potential physiological monothiol cosubstrate, was used in outer ring deiodination assays. These results demonstrate that thiol cosubstrate interactions with C124 in type 1 deiodinase play an important role in enhancing catalytic efficiency for both outer and inner ring deiodination.

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The role of OXA-1 β-lactamase Asp66 in the stabilization of the active-site carbamate group and in substrate turnover

Biochemical Journal ◽

10.1042/bj20070573 ◽

2008 ◽

Vol 410 (3) ◽

pp. 455-462 ◽

Cited By ~ 15

Author(s):

David A. Leonard ◽

Andrea M. Hujer ◽

Brian A. Smith ◽

Kyle D. Schneider ◽

Christopher R. Bethel ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Fold Increase ◽

Luria Bertani ◽

Saturation Mutagenesis ◽

Substrate Affinity ◽

Luria Bertani Broth ◽

Class D ◽

Carbamate Group

The OXA-1 β-lactamase is one of the few class D enzymes that has an aspartate residue at position 66, a position that is proximal to the active-site residue Ser67. In class A β-lactamases, such as TEM-1 and SHV-1, residues adjacent to the active-site serine residue play a crucial role in inhibitor resistance and substrate selectivity. To probe the role of Asp66 in substrate affinity and catalysis, we performed site-saturation mutagenesis at this position. Ampicillin MIC (minimum inhibitory concentration) values for the full set of Asp66 mutants expressed in Escherichia coli DH10B ranged from ≤8 μg/ml for cysteine, proline and the basic amino acids to ≥256 μg/ml for asparagine, leucine and the wild-type aspartate. Replacement of aspartic acid by asparagine at position 66 also led to a moderate enhancement of extended-spectrum cephalosporin resistance. OXA-1 shares with other class D enzymes a carboxylated residue, Lys70, that acts as a general base in the catalytic mechanism. The addition of 25 mM bicarbonate to Luria–Bertani-broth agar resulted in a ≥16-fold increase in MICs for most OXA-1 variants with amino acid replacements at position 66 when expressed in E. coli. Because Asp66 forms hydrogen bonds with several other residues in the OXA-1 active site, we propose that this residue plays a role in stabilizing the CO2 bound to Lys70 and thereby profoundly affects substrate turnover.

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Ionic homeostasis and subcellular element compartmentation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010013434x ◽

1990 ◽

Vol 48 (2) ◽

pp. 150-151

Author(s):

Ann LeFurgey ◽

Peter Ingram ◽

J.J. Blum ◽

M.C. Carney ◽

L.A. Hawkey ◽

...

Keyword(s):

Leishmania Major ◽

Ionic Homeostasis ◽

X Ray ◽

Functional Studies ◽

Cell Compartments ◽

X Ray Imaging ◽

Resolution Electron ◽

Multiple Cell ◽

Role Of Zn

Subcellular compartments commonly identified and analyzed by high resolution electron probe x-ray microanalysis (EPXMA) include mitochondria, cytoplasm and endoplasmic or sarcoplasmic reticulum. These organelles and cell regions are of primary importance in regulation of cell ionic homeostasis. Correlative structural-functional studies, based on the static probe method of EPXMA combined with biochemical and electrophysiological techniques, have focused on the role of these organelles, for example, in maintaining cell calcium homeostasis or in control of excitation-contraction coupling. New methods of real time quantitative x-ray imaging permit simultaneous examination of multiple cell compartments, especially those areas for which both membrane transport properties and element content are less well defined, e.g. nuclei including euchromatin and heterochromatin, lysosomes, mucous granules, storage vacuoles, microvilli. Investigations currently in progress have examined the role of Zn-containing polyphosphate vacuoles in the metabolism of Leishmania major, the distribution of Na, K, S and other elements during anoxia in kidney cell nuclel and lysosomes; the content and distribution of S and Ca in mucous granules of cystic fibrosis (CF) nasal epithelia; the uptake of cationic probes by mltochondria in cultured heart ceils; and the junctional sarcoplasmic retlculum (JSR) in frog skeletal muscle.

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