The flavoenzyme azobenzene reductase AzoR from Escherichia coli binds roseoflavin mononucleotide (RoFMN) with high affinity and is less active in its RoFMN form

Article abstract of DOI:10.1021/bi400348d

The Gram-positive bacterium Streptomyces davawensis is the only organism known to produce the antibiotic roseoflavin. Roseoflavin is a structural riboflavin analogue and is converted to the flavin mononucleotide (FMN) analogue roseoflavin mononucleotide (RoFMN) by flavokinase. FMN-dependent homodimeric azobenzene reductase (AzoR) (EC 1.7.1.6) from Escherichia coli was analyzed as a model enzyme. In vivo and in vitro experiments revealed that RoFMN binds to the AzoR apoenzyme with an even higher affinity compared to that of the "natural" cofactor FMN. Structural analysis (at a resolution of 1.07 A) revealed that RoFMN binding did not affect the overall topology of the enzyme and also did not interfere with dimerization of AzoR. The AzoR-RoFMN holoenzyme complex was found to be less active (30% of AzoR-FMN activity) in a standard assay. We provide evidence that the different physicochemical properties of RoFMN are responsible for its reduced cofactor activity.

Full text of DOI:10.1021/bi400348d

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Subscriber access provided by Purdue University Libraries
Article
THE FLAVOENZYME AZOBENZENE REDUCTASE AZOR FROM
ESCHERICHIA COLI BINDS ROSEOFLAVIN MONONUCLEOTIDE (ROFMN)
WITH HIGH AFFINITY AND IS LESS ACTIVE IN ITS ROFMN FORM
Simone Langer, Shinobu Nakanishi, Tilo Mathes, Tanja Knaus, Alexandra Binter, Peter
Macheroux, Tomoko Mase, Takuya Miyakawa, Masaru Tanokura, and Matthias Mack
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi400348d • Publication Date (Web): 28 May 2013
Downloaded from http://pubs.acs.org on May 31, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street
N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 1 of 34
Biochemistry
Flavin analogs as antibiotics
THE FLAVOENZYME AZOBENZENE REDUCTASE AZOR FROM
1
2
3
4
5
6
7
8
9
ESCHERICHIA COLI BINDS ROSEOFLAVIN MONONUCLEOTIDE
(ROFMN) WITH HIGH AFFINITY AND IS LESS ACTIVE IN ITS ROFMN
FORM*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Simone Langer1‡, Shinobu Nakanishi1‡, Tilo Mathes2, Tanja Knaus3, Alexandra
Binter3, Peter Macheroux3, Tomoko Mase4, Takuya Miyakawa4, Masaru
Tanokura4, and Matthias Mack1
1Department of Biotechnology, Institute for Technical Microbiology, Mannheim
University of Applied Sciences, 68163 Mannheim, Germany
2Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin,
10115 Berlin, Germany
3Institute of Biochemistry, Graz University of Technology, 8010 Graz, Austria
4Department of Applied Biological Chemistry, Graduate School of Agricultural and
Life Sciences, University of Tokyo, Tokyo 113ꢀ8657, Japan
*Running title: Flavin analogs as antibiotics.
To whom correspondence should be addressed: Matthias Mack, Institute for Technical
Microbiology, Mannheim University of Applied Sciences, PaulꢀWittsackꢀStr. 10,
68163 Mannheim, Germany, Tel.:+49ꢀ621ꢀ292ꢀ6496; Fax:+49ꢀ621ꢀ292ꢀ6420; Eꢀmail:
m.mack@hsꢀmannheim.de
‡These individuals contributed equally to this work.
1
ACS Paragon Plus Environment

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Biochemistry
Page 2 of 34
Flavin analogs as antibiotics
1
2
3
4
5
Keywords: Roseoflavin, Streptomyces davawensis, flavoenzymes, azobenzene
reductase, Escherichia coli
6
7
8
9
*This work was funded by the German “Federal Ministry of Education and Research”
(BMBF) (“Qualifizierungsꢀ/Profilierungsgruppe neue Technologien“). TM was
supported by the German Research Foundation (DFG).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 3 of 34
Biochemistry
Flavin analogs as antibiotics
1
2
3
4
ABSTRACT
5
6
7
8
The Gramꢀpositive bacterium Streptomyces davawensis is the only organism
known to produce the antibiotic roseoflavin. Roseoflavin is a structural riboflavin
analog and is converted to the flavin mononucleotide (FMN) analog roseoflavin
mononucleotide (RoFMN) by flavokinase. FMN dependent homodimeric azobenzene
reductase (AzoR) (EC 1.7.1.6) from Escherichia coli was analyzed as a model
enzyme. In vivo and in vitro experiments revealed that RoFMN binds to AzoR
apoenzyme with an even higher affinity when compared to the “natural” cofactor
FMN. Structural analysis (at a resolution of 1.07 Å) revealed that RoFMN binding did
not affect the overall topology of the enzyme and also did not interfere with
dimerization of AzoR. AzoRꢀRoFMN holoenzyme was found to be less active (30%
of AzoRꢀFMN activity) in a standard assay. We provide evidence that the different
physicochemical properties of RoFMN are responsible for its reduced cofactor
activity.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In all organisms riboflavin (vitamin B2) serves as the precursor for the flavoprotein
cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (1).
FMN is synthesized from riboflavin and ATP by flavokinase (EC 2.7.1.26), whereas
FAD is synthesized from FMN and ATP by FAD synthetase (2.7.7.2) (2). The
antibiotic roseoflavin (8ꢀdimethylaminoꢀ8ꢀdemethylꢀriboflavin) is produced by the
Gramꢀpositive bacterium Streptomyces davawensis (3, 4). Roseoflavin is toxic to
Gramꢀpositive but also to Gramꢀnegative bacteria if roseoflavin is able to enter the
cell (4-7). Roseoflavin was reported to have a profound inhibiting effect on the
growth and infectivity of the human bacterial pathogen Listeria monocytogenes at
very low concentrations (8). Many organisms contain riboflavin uptake systems which
3
ACS Paragon Plus Environment