Significance of individual amino acid residues for coenzyme and substrate specificity of 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus

We reported recently that regulation by intracellular pH (pHi) of the murine Cl−/HCO3− exchanger AE2 requires amino acid residues 310–347 of the polypeptide's NH2-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl− efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pHo) with unclamped or clamped pHi, or during variation of pHi at constant pHo. Wild-type AE2–mediated 36Cl− efflux was profoundly inhibited by acid pHo, with a value of pHo(50) = 6.87 ± 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312–347 identified the greatest acid shift in pHo(50) value, ∼0.8 pH units in the mutant (A)6342–347, but only a modest acid-shift in the mutant (A)6336–341. Two of the six (A)6 mutants retained normal pHi sensitivity of 36Cl− efflux, whereas the (A)6 mutants 318–323, 336–341, and 342–347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336–347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pHo and to pHi were found independently and in concert. The E346A mutation acid-shifted the pHo(50) value to the same extent whether pHi was unclamped or held constant during variation of pHo. Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pHo as well as pHi.

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Identification of Amino Acid Residues of the Retroviral Aspartic Proteinases Important for Substrate Specificity and Catalytic Efficiency

Aspartic Proteinases - Advances in Experimental Medicine and Biology ◽

10.1007/978-1-4615-1871-6_52 ◽

1995 ◽

pp. 399-406 ◽

Cited By ~ 1

Author(s):

C. E. Cameron ◽

H. Burstein ◽

D. Bizub-Bender ◽

T. Ridky ◽

I. T. Weber ◽

...

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Catalytic Efficiency ◽

Amino Acid Residues ◽

Aspartic Proteinases

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Prediction of amino acid residues participated in substrate recognition by cytochrome P450 subfamilies with broad substrate specificity

Journal of Molecular Recognition ◽

10.1002/jmr.2251 ◽

2013 ◽

Vol 26 (2) ◽

pp. 86-91 ◽

Cited By ~ 5

Author(s):

Maria S. Zharkova ◽

Boris N. Sobolev ◽

Nina Yu. Oparina ◽

Alexander V. Veselovsky ◽

Alexander I. Archakov

Keyword(s):

Cytochrome P450 ◽

Amino Acid ◽

Substrate Specificity ◽

Substrate Recognition ◽

Amino Acid Residues ◽

Broad Substrate Specificity

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Identification of the Substrate Specificity-conferring Amino Acid Residues of 4-Coumarate:Coenzyme A Ligase Allows the Rational Design of Mutant Enzymes with New Catalytic Properties

Journal of Biological Chemistry ◽

10.1074/jbc.m100355200 ◽

2001 ◽

Vol 276 (29) ◽

pp. 26893-26897 ◽

Cited By ~ 47

Author(s):

Hans-Peter Stuible ◽

Erich Kombrink

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Rational Design ◽

Catalytic Properties ◽

Amino Acid Residues

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Gene Cloning and Molecular Characterization of Lysine Decarboxylase from Selenomonas ruminantiumDelineate Its Evolutionary Relationship to Ornithine Decarboxylases from Eukaryotes

Journal of Bacteriology ◽

10.1128/jb.182.23.6732-6741.2000 ◽

2000 ◽

Vol 182 (23) ◽

pp. 6732-6741 ◽

Cited By ~ 38

Author(s):

Yumiko Takatsuka ◽

Yoshihiro Yamaguchi ◽

Minenobu Ono ◽

Yoshiyuki Kamio

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Pyridoxal Phosphate ◽

Evolutionary Relationship ◽

Binding Domain ◽

Sequencing Analysis ◽

Lysine Decarboxylase ◽

Amino Acid Residues ◽

Phosphate Binding ◽

Link Type

ABSTRACT Lysine decarboxylase (LDC; EC 4.1.1.18 ) from Selenomonas ruminantium comprises two identical monomeric subunits of 43 kDa and has decarboxylating activities toward both l-lysine andl-ornithine with similar Km andVmax values (Y. Takatsuka, M. Onoda, T. Sugiyama, K. Muramoto, T. Tomita, and Y. Kamio, Biosci. Biotechnol. Biochem. 62:1063–1069, 1999). Here, the LDC-encoding gene (ldc) of this bacterium was cloned and characterized. DNA sequencing analysis revealed that the amino acid sequence of S. ruminantium LDC is 35% identical to those of eukaryotic ornithine decarboxylases (ODCs; EC 4.1.1.17 ), including the mouse,Saccharomyces cerevisiae, Neurospora crassa,Trypanosoma brucei, and Caenorhabditis elegansenzymes. In addition, 26 amino acid residues, K69, D88, E94, D134, R154, K169, H197, D233, G235, G236, G237, F238, E274, G276, R277, Y278, K294, Y323, Y331, D332, C360, D361, D364, G387, Y389, and F397 (mouse ODC numbering), all of which are implicated in the formation of the pyridoxal phosphate-binding domain and the substrate-binding domain and in dimer stabilization with the eukaryotic ODCs, were also conserved inS. ruminantium LDC. Computer analysis of the putative secondary structure of S. ruminantium LDC showed that it is approximately 70% identical to that of mouse ODC. We identified five amino acid residues, A44, G45, V46, P54, and S322, within the LDC catalytic domain that confer decarboxylase activities toward bothl-lysine and l-ornithine with a substrate specificity ratio of 0.83 (defined as thek cat/Km ratio obtained with l-ornithine relative to that obtained withl-lysine). We have succeeded in converting S. ruminantium LDC to form with a substrate specificity ratio of 58 (70 times that of wild-type LDC) by constructing a mutant protein, A44V/G45T/V46P/P54D/S322A. In this study, we also showed that G350 is a crucial residue for stabilization of the dimer in S. ruminantium LDC.

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