%PDF-1.4
%
16 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
10 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
4 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
7 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
1 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
3 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
13 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
14 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
6 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
11 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
9 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
8 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
12 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
5 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
15 0 obj
<>>>/BBox[0 0 584.96 782.99]/Length 132>>stream
x5ͱ0OqFmoQ7@5-pWjQbgapT`,yv-$òQQiS#Iz]`[HaabfrjG[?J]J/&
endstream
endobj
18 0 obj
<>stream
Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45
10.1128/aem.00029-22
Leanne P. Sims
Colin W. J. Lockwood
Andrew T. Crombie
Justin M. Bradley
Nick E. Le Brun
J. Colin Murrell
Enzymology and Protein Engineering
Enzymology and Protein Engineering
Actinobacteria
soluble diiron monooxygenase
isoprene metabolism
volatile organic compounds
American Society for Microbiology
Sims et al.
Copyright © 2022 Sims et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
20220314
2022
ABSTRACT
Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene.
IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.
20220110
20220130
1098-5336
Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version)2022-03-23T06:07:07-07:00
endstream
endobj
19 0 obj
<>stream
x+ |
endstream
endobj
20 0 obj
<>stream
xS**T0T0 Bi yJ%
endstream
endobj
21 0 obj
<>stream
x+ |
endstream
endobj
22 0 obj
<>stream
xS**T0T0 Bi yS&
endstream
endobj
23 0 obj
<>stream
x+ |
endstream
endobj
24 0 obj
<>stream
xS**T0T0 Bi yn)
endstream
endobj
25 0 obj
<>stream
x+ |
endstream
endobj
26 0 obj
<>stream
xS**T0T0 Bi yA$
endstream
endobj
27 0 obj
<>stream
x+ |
endstream
endobj
28 0 obj
<>stream
xS**T0T0 Bi y\'
endstream
endobj
29 0 obj
<>stream
x+ |
endstream
endobj
30 0 obj
<>stream
xS**T0T0 Bih uU
endstream
endobj
31 0 obj
<>stream
x+ |
endstream
endobj
32 0 obj
<>stream
xS**T0T0 Bih ~V
endstream
endobj
33 0 obj
<>stream
x+ |
endstream
endobj
34 0 obj
<>stream
xS**T0T0 Bih X
endstream
endobj
35 0 obj
<>stream
x+ |
endstream
endobj
36 0 obj
<>stream
xS**T0T0 Bi y8#
endstream
endobj
37 0 obj
<>stream
x+ |
endstream
endobj
38 0 obj
<>stream
xS**T0T0 Bih W
endstream
endobj
39 0 obj
<>stream
x+ |
endstream
endobj
40 0 obj
<>stream
xS**T0T0 Bih lT
endstream
endobj
41 0 obj
<>stream
x+ |
endstream
endobj
42 0 obj
<>stream
xS**T0T0 Bi y+
endstream
endobj
43 0 obj
<>stream
x+ |
endstream
endobj
44 0 obj
<>stream
xS**T0T0 Bi y,
endstream
endobj
45 0 obj
<>stream
x+ |
endstream
endobj
46 0 obj
<>stream
xS**T0T0 Bi ye(
endstream
endobj
47 0 obj
<>stream
x+ |
endstream
endobj
48 0 obj
<>stream
xS**T0T0 Bi yw*
endstream
endobj
50 0 obj
<>stream
HW]sۺW1@iNo:sK%HJRqro삒(;M'3D <{j5zs;w_UW^dYeLWZ5KӼEa~c-l[14kmzU^c:PVXUnk/"mvk|Ҵ4~s&hA*IT-rkȈ_]TT
i[dKEכO՛!`m*(ЮzG*Uiah?NN^XK4u
hv_YfrѤJ%iuǞ)a7k ) ]QP@TUnpwЎ$>9BP7[gdzXve"H^KX˅KquE~5Iĸ"H^TchËkujV.}7^GW F#D-qtv,i5ːC)so$M}
jQ%"݅~IX+T
/Mp:ƘQ