ZOUHAR, Jan, Jitka VÉVODOVÁ, Jaromír MAREK, Jiří DAMBORSKÝ, X.-D. SU and Břetislav BRZOBOHATÝ. Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1. Plant Physiology. USA: American Society of Plant Physiologists, 2001, vol. 127, No 3, p. 973-985. ISSN 0032-0889.
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Basic information
Original name Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1
Authors ZOUHAR, Jan (203 Czech Republic, belonging to the institution), Jitka VÉVODOVÁ (203 Czech Republic, belonging to the institution), Jaromír MAREK (203 Czech Republic, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, belonging to the institution), X.-D. SU and Břetislav BRZOBOHATÝ (203 Czech Republic, guarantor, belonging to the institution).
Edition Plant Physiology, USA, American Society of Plant Physiologists, 2001, 0032-0889.
Other information
Original language English
Type of outcome Article in a journal
Field of Study Genetics and molecular biology
Country of publisher United States of America
Confidentiality degree is not subject to a state or trade secret
Impact factor Impact factor: 5.105
RIV identification code RIV/00216224:14310/01:00004635
Organization unit Faculty of Science
UT WoS 000172251200029
Keywords in English crystal structure
Changed by Changed by: doc. RNDr. Jaromír Marek, Ph.D., učo 1989. Changed: 2/5/2012 13:16.
Abstract
The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased Km of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in kcat was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.
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MSM 143100005, plan (intention)Name: Strukturně-funkční vztahy biomolekul a jejich role v metabolismu
Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Structure-function Relationships and their role in the Metabolism
MSM 143100008, plan (intention)Name: Genomy a jejich funkce
Investor: Ministry of Education, Youth and Sports of the CR, Genomes and their functions
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