Insights into the Functional Architecture of the Catalytic
Center of a Maize Beta-Glucosidase Zm-p60.1

J 2001

Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1

ZOUHAR, Jan, Jitka VÉVODOVÁ, Jaromír MAREK, Jiří DAMBORSKÝ, X.-D. SU et. al.

Základní údaje

Originální název

Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1

Autoři

ZOUHAR, Jan (203 Česká republika, domácí), Jitka VÉVODOVÁ (203 Česká republika, domácí), Jaromír MAREK (203 Česká republika, domácí), Jiří DAMBORSKÝ (203 Česká republika, domácí), X.-D. SU a Břetislav BRZOBOHATÝ (203 Česká republika, garant, domácí)

Vydání

Plant Physiology, USA, American Society of Plant Physiologists, 2001, 0032-0889

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 5.105

Kód RIV

RIV/00216224:14310/01:00004635

Organizační jednotka

Přírodovědecká fakulta

UT WoS

000172251200029

Klíčová slova anglicky

crystal structure

Změněno: 2. 5. 2012 13:16, doc. RNDr. Jaromír Marek, Ph.D.

Anotace

V originále

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.

Návaznosti

MSM 143100005, záměr

Název: Strukturně-funkční vztahy biomolekul a jejich role v metabolismu

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Strukturně-funkční vztahy biomolekul a jejich role v metabolismu

MSM 143100008, záměr

Název: Genomy a jejich funkce

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Genomy a jejich funkce

Citovat

ZOUHAR, Jan, Jitka VÉVODOVÁ, Jaromír MAREK, Jiří DAMBORSKÝ, X.-D. SU a 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, roč. 127, č. 3, s. 973-985. ISSN 0032-0889.

@article{375120,
   author = {Zouhar, Jan and Vévodová, Jitka and Marek, Jaromír and Damborský, Jiří and Su, X.andD. and Brzobohatý, Břetislav},
   article_location = {USA},
   article_number = {3},
   keywords = {crystal structure},
   language = {eng},
   issn = {0032-0889},
   journal = {Plant Physiology},
   title = {Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1},
   volume = {127},
   year = {2001}
}

TY  - JOUR
ID  - 375120
AU  - Zouhar, Jan - Vévodová, Jitka - Marek, Jaromír - Damborský, Jiří - Su, X.-D. - Brzobohatý, Břetislav
PY  - 2001
TI  - Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1
JF  - Plant Physiology
VL  - 127
IS  - 3
SP  - 973
EP  - 973
PB  - American Society of Plant Physiologists
SN  - 00320889
KW  - crystal structure
N2  - 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.
ER  -

ZOUHAR, Jan, Jitka VÉVODOVÁ, Jaromír MAREK, Jiří DAMBORSKÝ, X.-D. SU a Břetislav BRZOBOHATÝ. Insights into the Functional Architecture of the Catalytic Center of a Maize Beta-Glucosidase Zm-p60.1. \textit{Plant Physiology}. USA: American Society of Plant Physiologists, 2001, roč.~127, č.~3, s.~973-985. ISSN~0032-0889.

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