Supporting Information for
Structural and Biochemical Characterization of the
Cytochrome P450 CypX (CYP134A1) from Bacillus
subtilis: cyclo-L-leucyl-L-leucyl Dipeptide Oxidase
Max J. Cryle1†*, Stephen G. Bell2 and Ilme Schlichting1
1
Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29,
69120 Heidelberg, Germany
2
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road,
Oxford OX1 3QR, UK
Max.Cryle@mpimf-heidelberg.mpg.de
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
* Address correspondence to: Max J. Cryle, Max-Planck Institute for Medical Research,
Jahnstrasse 29, 69120 Heidelberg, Germany. Telephone: +49 (6221) 486 516; Fax: +49 (6221) 486 585;
E-mail:Max.Cryle@mpimf-heidelberg.mpg.de
1
SI Figure 1. Binding curves for substrates cLL (A), cVV (B), cMM (C), cLF (D), 2,5-di-t-butylquinone
(E) and 2,5-di-t-butylhydroxyquinone (F) to CYP134A1.
2
SI Table 1. Retention times and MS fragmentation of cLL, cLF and cMM together with compounds
present in the GCMS analysis of the CYP134A1 turnover experiments.
Compound
Retention Time
MS fragmentation a
cLL
20.72 minutes
m/z 211 (2.2%), 183 (6.4%), 171 (9.6%), 170
(100%), 155 (5.4%), 140 (35.7%), 114
(46.8%), 86 (97.6%)
cLL
monomethylated 19.88 minutes
phenol tautomer b
m/z 197 (3.0%), 185 (10.8%), 184 (100%),
155 (51.0%), 128 (52.5%), 127 (67.2%), 100
(38.3%), 86 (38.3%)
monooxygenated
monomethylated
tautomer b
cLL 19.97 minutes
phenol
m/z 200 (23.9%), 171 (12.8%), 170 (10.1%),
169 (28.3%), 141 (8.6%), 117 (7.3%), 116
(100%), 86 (32.9%)
28.41 minutes
m/z 260 (9.9%), 217 (2.2%), 204 (45.5%),
169 (19.0%), 141 (36.5%), 120 (13.1%), 113
(44.1%), 91 (100%)
cLF
monomethylated 27.58 minutes
phenol tautomer b
m/z 274 (13.3%), 231 (0.3%), 218 (34.6%),
183 (76.1%), 155 (100%), 134 (0.6%), 127
(83.9%), 91 (26.5%)
cLF dimethylated phenol 26.74 minutes
tautomer b
m/z 288 (5.2%), 232 (21.5%), 197 (39.6%),
169 (25.8%), 141 (100%), 113 (43.8%), 91
(11.7%)
monooxygenated
monomethylated
tautomer b
cLF 27.63 minutes
phenol
m/z 259 (1.4%), 234 (6.5%), 218 (83.4%),
203 (5.6%), 189 (23.2%), 127 (94.7%), 99
(83.2%), 91 (100%)
31.60 minutes
m/z 262 (33.9%), 201 (18.4%), 188 (57.4%),
173 (11.1%), 153 (21.6%), 140 (28.3%), 114
(100%), 61 (74.5%)
cMM
monomethylated 30.71 minutes
phenol tautomer b
m/z 276 (20.2%), 215 (12.4%), 202 (51.2%),
187 (11.0%), 167 (11.4%), 154 (11.0%), 128
(100%), 61 (47.4%)
cMM dimethylated phenol 29.73 minutes
tautomer b
m/z 290 (17.0%), 229 (9.8%), 216 (36.9%),
201 (3.4%), 181 (5.4%), 155 (18.2%), 142
(100%), 61 (29.0%)
cMM aromatized phenol 27.29 minutes
tautomer b
m/z 214 (42.7%), 199 (6.8%), 153 (93.2%),
140 (100%), 125 (16.6%), 112 (62.7%), 95
(24.9%), 61 (58.6%)
cLF
cMM
a
MS peak followed by relative intensity
b
Identified on the basis of MS fragmentation, see main text.
3
4
SI Figure 2. GC-traces of CYP134A1-mediated substrate turnover using the the Pux/PuR redox system.
A – top trace: cLL oxidation; A – lower trace: comparison of cLL oxidation (pink trace, upper) to cLL
blank excluding NADH (black trace, lower); B – cLF oxidation (product identified using m/z 234, pink
trace); C – cMM oxidation.
5
SI Table 2. Bacillus subtilis P450s.
P450
CYP107H1
BioI
Function
PDB Code
Biotin Operon (1)
3EJB
Forms pimelic acid via carbon-carbon 3EJD
bond cleavage of fatty acyl-ACPs (2, 3)
3EJE
CYP102A2
CypD
Redox partner fusion proteins
CYP102A3
CypE
Fatty acid hydroxylase activity (4-6)
CYP107K1
PksS
Bacillaene Metabolism (7)
CYP134A1
CypX
Pulcheriminic Acid Biosynthesis
This study
cLL oxidase
CYP109B1
YjiB
Unknown function
41% identity, 60% Similarity to P450meg
(Steroid Hydroxylase, CYP106A2)
CYP107J1
CypA
Unknown function
CYP152A1
Bsβ
Fatty acid α/β hydroxylase
2ZQJ
Utilizing hydrogen peroxide (8, 9)
2ZQX
Bacillus subtilis contains eight P450s, several of which have been characterized and have shown to
catalyse unusual transformations (SI Table 2). These include CYP107H1, found in the biotin operon of
B. subtilis, which is responsible for the biosynthesis of pimelic acid, a seven-carbon diacid precursor of
biotin, from long chain fatty acids (1, 3). The substrates for CYP107H1 are fatty acyl-ACPs (acyl carrier
protein) and the enzyme catalyses the oxidation of the C7 and C8 carbons of the chain, followed by
cleavage of the intervening C-C bond (10). This enzyme has also been shown to be active in forming
pimelic acid and hydroxylated fatty acids from free fatty acids in vitro (11). A further fatty acid
hydroxylase, CYP152A has been shown to oxidize the α- and β-positions of fatty acids. This enzyme
requires hydrogen peroxide for function, unlike the vast majority of P450s that require electrons from an
electron transport ultimately derived from NAD(P)H (8, 9).
6
SI Table 3. Structural comparisons of known P450 structures with similarity to CYP134A1.a
P450
PDB Entry
Percentage
Sequence
Identity (%)
RMSD
(Å)
No. Aligned Residues/ Z-score
Total Residues
(DALI)
CalO2
3BUJ
27
2.2
360/ 397
43.0
26
2.3
364/ 397
42.6
26
2.0
352/ 385
42.5
26
2.0
355/ 385
42.3
26
2.2
355/ 403
41.1
27
2.2
360/ 403
41.1
26
2.1
357/ 403
40.6
26
2.2
362/ 403
40.2
26
2.3
355/ 393
40.1
26
2.3
359/ 393
40.2
23
2.3
349/ 394
37.9
23
2.5
351/ 394
36.7
BioI
EryF
EpoK
EryK
CYP121 b
3EJB
1OXA
1PKF
2WIO
3G5H
a
First set of values are compared to Native-1 structure; second set of values are compared to Native-2
structure.
b
Included as CYP121 is the only other P450 known to oxidise a cyclic dipeptide.
7
Sequence Comparison of CYP134A1 with Closely Related P450s. A BLAST search and sequence
analysis of the closest sequence matches to CYP134A1 from Bacillus subtilis indicate that the closest
matches could be divided essentially into three groups on the basis of conservation of sequence motifs
(see SI Tables 4 and 5). The first group, which contains CYP134A1 from B. subtilis, includes ten P450s
by and large annotated as “CypX” and that possess similar sequences, with a very high degree of
sequence conservation in the P450 substrate recognition sites (SRSs) (SI Table 4). This conservation
includes the unusual proline residue Pro-237 that replaces the typically conserved alcohol I-helix residue
involved in oxygen activation. The high degree of conserved SRS residues in this group of P450s
confers active sites essentially identical across the relevant portions of the F-G-helices, β1-sheet, I-helix
and C-terminal loop. The B-B2 loop region is rather more variable, although there is a core of residues
(64, 67-72) that are highly conserved across all sequences. Interestingly, this includes the residues Leu64 and Arg-67 that are observed in the crystal structures to project into the active site in the “closed”
conformation of this loop, supporting a potential role for such a structure in solution. With one
exception (that of CypX from Streptomyces sp. Mg1), these P450s also have an upstream cyclic
dipeptide synthase gene, the majority of which have been shown to produce cLL with relatively high
selectivity (12). When combined with the data obtained for CYP134A1 from B. subtilis, it can be
postulated that these P450s all catalyse the same oxidation on cLL substrates. Additionally, all these
P450 possess an extended loop on the proximal heme face prior to the heme-ligating cysteine residue,
implying a similar role for this structural element across the different organisms possessing these P450s.
The second group of P450s with similarity to CYP134A1 include predominantly P450s from various
species of Mycobacterium, and include the Mycobacterium tuberculosis P450 CYP121 (SI Table 5,
Group 2) (13). This P450, the only other P450 to date shown to oxidize cyclic dipeptides, catalyses the
phenolic coupling of the tyrosine aromatic ring moieties of cYY (14). The presence of an upstream
cyclic dipeptide synthase is only found in the case of CYP121, with the exact role of the other P450s
unclear. This group of P450s display a different set of catalytic residues in the I-helix (the typical acid/
8
alcohol pairing), in addition to alterations of various SRS motifs. Moreover, the C-terminal loop
extension found in CYP134A1 is not present in this group of P450s.
The third group of P450s with similarity to CYP134A1 are mostly found in various Streptomyces
species (SI Table 5, Group 3). These P450s do not have upstream CDPS genes and do not contain the
unusual active site residues found in the first group of P450s, indicating a different substrate and
possible mechanism to that seen for CYP134A1. The C-terminal loop extension is also found in this
group, inferring that the role of this loop is not specific to the CYP134A1 oxidative mechanism. Rather,
it would appear that this extension plays a role in moderating the redox interactions of the P450 with
endogenous redox partner proteins.
9
SI Table 4. Substrate recognition site (SRS) alignment of P450s (Group 1) with high SRS homology to
CYP134A1.
Species / P450 a
B-B2 loop
FG-helices
I-helix
b
β1-sheet
332-341
Insertion
Loop
391
/
c
392
O34926
FTTKSLVERAEPVMRGPVLAQMH
VADFITSISQ
AATEPADKT
PVQLIPR
GIKSAFSGAAR
YT
FTTKSLAKRAEPVMRGPVLAQMK
VADFITSLNQ
AATEPADKT
PVQLIPR
EVKSAFSGAAR
YT
FTTKSLAERAEPVMRGPVLAQMR
VADFITSINQ
AATEPGDKT
PVQLIPR
DIKKAFSGAAR
YT
FTTKSLAERAEPVMRGPVLAQMR
VADFITSINQ
AATEPGDKT
PVQLIPR
DIKKAFSGAAR
YT
FTTKSLAERAEPVMRGPVLAQMR
VAEFITSINQ
AATEPGDKT
PVQLIPR
DIKKAFSGAAR
YT
B. cereus
FTTKSLAERAEPVMRGPVLAQMR
VADFITSINQ
AATEPGDKT
PVQLIPR
DIKKAFSGAAR
YT
B4UYE5
FTTETLQVRAEPVMRGPVLAQMT
VAEFITSLDL
AATEPADKT
PVQLIPR
GTARSFTAAAQ
YT
S.
FTTKTLAERAEPVMKDRVLAQMS
IAKFITSFNL
AATEPVDKT
PVQLIPR
ESNKPFTSHSQ
YT
P.
FNTKPLTALAEPVMGDRVLAQME
IASFITQFDQ
AATEPADKI
PVQLIPR
TTSPQKANRKR
YT
C.
FSTDHLATRAEPVLGDRVLAQMT
IVKFITLLQQ
AASEPLDKT
PVQIIPR
VPRSAFTPSAK
YT
*.*. *
:..*** :.
**:** **
***:***
Bacillus subtilis
Q65EX2
B.licheniformis
Q3F0K6
B.
thuringiensis
(ATCC 35646)
C3IVY8
B.thuringiensis
(IBL 4222)
C3CBX1
B.
thuringiensis
(BGSC 4Y1)
Streptomyces sp.
Mg1
Q4L2X8
haemolyticus
Q7N9M6
luminescens
Q4JVR9
jeikeium
****: . *****
:
a
UniProtKB/TrEMBL Code
b
Residues in the position of the typically conserved acid/ alcohol residues emboldened.
:
**
c
Loop regions do not share the same degree as the SRSs, however this loop region is absent in the
majority of other P450 sequences.
10
SI Table 5. P450s with homology to CYP134A1 but significant alterations in critical structural
elements.
Group 2 P450s: 332-341 loop is absent and Group 3 P450s: 332-341 loop is present but
no active site proline residue
no active site proline residue
ID a
Organism
ID a
Organism
P0A514
Mycobacterium tuberculosis
Q0RU65
Frankia alni
Q08U53
Stigmatella aurantiaca
D1VL05
Frankia sp.
Q1B8YS
Mycobacterium sp. MCS
B5H3V0
Streptomyces clavuligerus
A1UG27
Mycobacterium sp. KMS
B5H7R3
Streptomyces pristinaespiralis
A0CQ81
Mycobacterium avium
Q9RJQ7
Streptomyces coelicolor
Q73WL1
Mycobacterium
paratuberculosis
A0ACY9
Streptomyces ambofaciens
Q83YE9
Streptomyces
hygroscopicus
subsp. yingchengensis
B1VUJ4
Streptomyces griseus subsp.
griseus
D1XAY7
Streptomyces sp. ACT-1
C9NFH8
Streptomyces flavogriseus
a
UniProtKB/TrEMBL Code
11
SI Figure 3. MS fragmentation of cLF (A) and the oxidized cLF product (B), showing the mass spectra
and the postulated fragmentation pathways for notable ions.
12
SI Figure 4. MS fragmentation of cMM (A) and the oxidized cMM product (B), showing the mass
spectra and the postulated fragmentation pathways for notable ions.
13
SI Figure 5. A – Optimal cLL docking solution obtained with the CYP134A1 in the open conformation
using Autodock (side chain geometry optimised), with hydrogen bonding interactions indicated to the
haem iron and Tyr-391; B – comparison of the position of the bound glycerol molecule and Tyr-391
residue in the CYP134A1 active site (Native 1, monomer A), with hydrogen bonding interactions
indicated to the haem iron and Tyr-391; C – overlayed structures of the docked cLL and bound glycerol
molecules (cLL shown in pink, cLL hydrogen-bonded Tyr-391 shown in grey, glycerol and hydrogenbonded glycerol shown in yellow, haem shown in red).
14
SI Figure 6. Optimal cLL docking solution obtained with the CYP134A1 in the open conformation
using Patchdock (side chain geometry not optimised), with hydrogen bonding interactions indicated to
the haem iron (cLL shown in green, haem shown in red).
15
SI Figure 7. Comparison of the surface charge on the proximal side of the haem group in various
Cytochrome P450s, with the view oriented along the central I-helix (CYP134A1 loop extension shown
16
in turquoise, protein shown in grey, haem shown in red; positive charge shown as a blue surface,
negative charge shown as a red surface). A – P450BM3 surface charge indicating the interaction interface
with the redox partner (15); B – P450cam surface charge indicating the computed interaction interface
with the redox partner (16); C and D – CYP134A1 surface charge including the 332 – 341 residue loop
extension (C) and with the 332 – 341 residue loop deleted (D), indicating that the loop is essentially
non-charged; E – the surface charge of CYP199A2, which utilises the PuX/ PuR redox system also
shown to sustain oxidation with CYP134A1 (17); F – the surface charge of P450BioI (F), which affords
product using the redox partner cindoxin, rather than PuX/ PuR type redox systems (3).
17
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18
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20