Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

Sang Choon Lee; Jinheung Kim; Im Joung La; Soon Kil Kim; Moon Young Yoon

doi:10.1016/j.enzmictec.2012.10.006

Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

Sang Choon Lee, Jinheung Kim, Im Joung La, Soon Kil Kim, Moon Young Yoon

Department of Chemistry & Nanoscience

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent K_m for pyruvate was calculated to be 1.37mM, while the K_c for thiamin diphosphate (ThDP) and Mg²⁺ were found to be 0.031μM and 1.27mM, respectively. Negligible absorbance at 450nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.

Original language	English
Pages (from-to)	54-59
Number of pages	6
Journal	Enzyme and Microbial Technology
Volume	52
Issue number	1
DOIs	https://doi.org/10.1016/j.enzmictec.2012.10.006
State	Published - 10 Jan 2013

Bibliographical note

Funding Information:
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( 2012R1A1A2008516 ) and by the “ Cooperative Research Program grants for Agriculture Science & Technology Development (Project No. PJ907052)” funded by the Rural Development Administration, Republic of Korea .

Keywords

Acetolactate synthase
Catabolic
Enterococcus faecalis
Flavin adenine dinucleotide
ThDP

Access to Document

10.1016/j.enzmictec.2012.10.006

Cite this

@article{c7deae81287f46a99096f7ebb38961f4,

title = "Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583",

abstract = "The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent Km for pyruvate was calculated to be 1.37mM, while the Kc for thiamin diphosphate (ThDP) and Mg2+ were found to be 0.031μM and 1.27mM, respectively. Negligible absorbance at 450nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.",

keywords = "Acetolactate synthase, Catabolic, Enterococcus faecalis, Flavin adenine dinucleotide, ThDP",

author = "Lee, {Sang Choon} and Jinheung Kim and La, {Im Joung} and Kim, {Soon Kil} and Yoon, {Moon Young}",

note = "Funding Information: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( 2012R1A1A2008516 ) and by the “ Cooperative Research Program grants for Agriculture Science & Technology Development (Project No. PJ907052)” funded by the Rural Development Administration, Republic of Korea . ",

year = "2013",

month = jan,

day = "10",

doi = "10.1016/j.enzmictec.2012.10.006",

language = "English",

volume = "52",

pages = "54--59",

journal = "Enzyme and Microbial Technology",

issn = "0141-0229",

publisher = "Elsevier Inc.",

number = "1",

}

TY - JOUR

T1 - Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

AU - Lee, Sang Choon

AU - Kim, Jinheung

AU - La, Im Joung

AU - Kim, Soon Kil

AU - Yoon, Moon Young

N1 - Funding Information: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( 2012R1A1A2008516 ) and by the “ Cooperative Research Program grants for Agriculture Science & Technology Development (Project No. PJ907052)” funded by the Rural Development Administration, Republic of Korea .

PY - 2013/1/10

Y1 - 2013/1/10

N2 - The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent Km for pyruvate was calculated to be 1.37mM, while the Kc for thiamin diphosphate (ThDP) and Mg2+ were found to be 0.031μM and 1.27mM, respectively. Negligible absorbance at 450nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.

AB - The catabolic acetolactate synthase (cALS) of Enterococcus faecalis V583 was cloned, expressed in Escherichia coli, and purified to homogeneity. The purified protein had a molecular weight of 60kDa. The cALS of E. faecalis is highly homologous with other cALSs, while sharing low homology with its anabolic counterparts. The cALS of E. faecalis exhibits optimum activity at a temperature of 37°C and pH 6.8. Based on the enzyme characterization, the apparent Km for pyruvate was calculated to be 1.37mM, while the Kc for thiamin diphosphate (ThDP) and Mg2+ were found to be 0.031μM and 1.27mM, respectively. Negligible absorbance at 450nm and lack of activity enhancement upon addition of flavin adenine dinucleotide (FAD) to the assay buffer suggest that the cALS of E. faecalis is not FAD-dependent. The enzyme showed extreme stability against the organic solvent dimethyl sulfoxide (DMSO), whereas the activity decreased to less than 50% in the presence of acetone and ethanol.

KW - Acetolactate synthase

KW - Catabolic

KW - Enterococcus faecalis

KW - Flavin adenine dinucleotide

KW - ThDP

UR - http://www.scopus.com/inward/record.url?scp=84870357325&partnerID=8YFLogxK

U2 - 10.1016/j.enzmictec.2012.10.006

DO - 10.1016/j.enzmictec.2012.10.006

M3 - Article

C2 - 23199739

AN - SCOPUS:84870357325

SN - 0141-0229

VL - 52

SP - 54

EP - 59

JO - Enzyme and Microbial Technology

JF - Enzyme and Microbial Technology

IS - 1

ER -

Characterization of recombinant FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583

Abstract

Bibliographical note

Keywords

Access to Document

Fingerprint

Cite this