Human Xanthine Oxidase Changes its Substrate Specificity to Aldehyde Oxidase Type upon Mutation of Amino Acid Residues in the Active Site: Roles of Active Site Residues in Binding and Activation of Purine Substrate

2007 ◽  
Vol 141 (4) ◽  
pp. 513-524 ◽  
Author(s):  
Y. Yamaguchi ◽  
T. Matsumura ◽  
K. Ichida ◽  
K. Okamoto ◽  
T. Nishino
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Xanthine Oxidase ◽  
Active Site ◽  
Aldehyde Oxidase ◽  
Amino Acid Residues ◽  
Active Site Residues

scholarly journals Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

2016 ◽  
Vol 60 (10) ◽  
pp. 6084-6090 ◽  
Author(s):  
Dandan He ◽  
Jiachi Chiou ◽  
Zhenling Zeng ◽  
Edward Wai-Chi Chan ◽  
Jian-Hua Liu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Structural Integrity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Significant Difference ◽  
Function Relationship ◽  
Alignment Analysis ◽  
M 14

ABSTRACTClinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between theblaCTX-M-15andblaCTX-M-14elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.

scholarly journals Site Directed Mutagenesis of Amino Acid Residues at the Active Site of Mouse Aldehyde Oxidase AOX1

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5348 ◽  
Author(s):  
Silvia Schumann ◽  
Mineko Terao ◽  
Enrico Garattini ◽  
Miguel Saggu ◽  
Friedhelm Lendzian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Aldehyde Oxidase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues

scholarly journals Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

2002 ◽  
Vol 76 (12) ◽  
pp. 5949-5958 ◽  
Author(s):  
Yuichi Someya ◽  
Naokazu Takeda ◽  
Tatsuo Miyamura
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Hepatitis A Virus ◽  
Side Chain ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
3C Protease ◽  
Catalytic Dyad ◽  
Positively Charged

ABSTRACT The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

scholarly journals Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhi-Zhong Song ◽  
Bin Peng ◽  
Zi-Xia Gu ◽  
Mei-Ling Tang ◽  
Bei Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Peach Fruit ◽  
Alcohol Acyltransferase

AbstractThe aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

Investigating the role of active site residues of Rhodotorula gracilis d-amino acid oxidase on its substrate specificity

Biochimie ◽  
2007 ◽  
Vol 89 (3) ◽  
pp. 360-368 ◽  
Author(s):  
Angelo Boselli ◽  
Luciano Piubelli ◽  
Gianluca Molla ◽  
Mirella S. Pilone ◽  
Loredano Pollegioni ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Active Site ◽  
Acid Oxidase ◽  
Active Site Residues ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

scholarly journals Identification of active-site residues of sheep liver serine hydroxymethyltransferase

1984 ◽  
Vol 224 (3) ◽  
pp. 703-707 ◽  
Author(s):  
R Manohar ◽  
N Appaji Rao
Keyword(s):  
Amino Acid ◽  
Chemical Modification ◽  
Active Site ◽  
Rate Constants ◽  
Second Order ◽  
Serine Hydroxymethyltransferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Diethyl Pyrocarbonate ◽  
Sheep Liver

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5′-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues†

Biochemistry ◽  
1998 ◽  
Vol 37 (41) ◽  
pp. 14605-14612 ◽  
Author(s):  
Gina J. Mancini-Samuelson ◽  
Volker Kieweg ◽  
Kim Marie Sabaj ◽  
Sandro Ghisla ◽  
Marian T. Stankovich
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Redox Properties ◽  
Amino Acid Residues ◽  
Medium Chain ◽  
Chain Acyl
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