Báo cáo Y học: Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system docx

Kinetic analysis of hydroxylation of saturated fatty acidssystem 7Tatsuya 7 Kitazume1, Akinori Tanaka1, Naoki Takaya1, Akira Nakamura1, Shigeru Matsuyama1, Takahisa Suzuki1and Hirofumi S

Kinetic analysis of hydroxylation of saturated fatty acids

system

7Tatsuya

7 Kitazume1, Akinori Tanaka1, Naoki Takaya1, Akira Nakamura1, Shigeru Matsuyama1,

Takahisa Suzuki1and Hirofumi Shoun2

1

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki, Japan;2Department of Biotechnology, Graduate School

of Agricultural and Life Sciences, The University of Tokyo, Japan

Cytochrome P450foxy (P450foxy, CYP505) is a fused

pro-tein of cytochrome P450 (P450) and its reductase isolated

from the fungus Fusarium oxysporum, which catalyzes the

subterminal (x-1
x-3) hydroxylation of fatty acids Here,

we produced, purified and characterized a fused

recombin-ant protein (rP450foxy) using the Escherichia coli expression

system Purified rP450foxy was catalytically and spectrally

indistinguishable from the native protein, but most of the

rP450foxy was recovered in the soluble fraction of E coli

cells unlike the membrane-bound native protein The results

are consistent with our notion that the native protein is

targeted to the membrane by a post-translational

modifica-tion mechanism We also discovered that P450foxy could use

shorter saturated fatty acid chains

sub-strate The regiospecificity (x-1
x-3) of hydroxylation due

to the enzymatic reaction for the short substrates (decanoate,

C10; undecanoate, C11) was the same as that for longer

substrates Steady state kinetic studies showed that the kcat values for all substrates tested (C9-C16) were of the same magnitude (1200–1800 min)1), whereas the catalytic effi-ciency (kcat/Km) was higher for longer fatty acids Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified Kinetic data were consistent with the inhibition due to binding of the second substrate We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding

Keywords: fatty acid hydroxylase; cytochrome P450; P450foxy; dodecanoic acid; Fusarium oxysporum

Cytochrome P450 (P450) is a group of heme proteins that

are widespread in nature [1–3] It is generally accepted that

all P450s originated from the same, ancient gene (P450

superfamily), which has acquired unparalleled molecular

and functional diversity during evolution [1,4] Most of the

P450 enzymes function as monooxygenases that act on

various lipophilic compounds, whereas others catalyze a

variety of reactions [2] P450s can be classified into several

classes according to their redox partners [5,6] Bacterial and

mitochondrial P450 systems are of class I; they receive

electrons from NAD(P)H via ferredoxin reductase and

ferredoxin coupling Eukaryotic microsomal P450 systems

are of class II; they receive electrons from P450 reductase,

which contains both FAD and FMN These two classes

comprise typical, multicomponent P450 monooxygenase

systems, whereas the functions of P450 are most diversified

in other classes Class III P450s are not monooxygenases

but catalyze isomerization [7] or dehydration [8], and require neither external redox equivalents nor any redox partners P450nor is the only class IV P450, and catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) using NAD(P)H as the direct electron donor [9] The class III and IV P450s are self-sufficient, meaning that they can complete their functions without other proteinaceous components

This laboratory has isolated two unique P450s from the fungus Fusarium oxysporum One is P450nor (CYP55) as described above [9–12], and the other is P450foxy (CYP505) [13,14], both of which are self-sufficient P450foxy is of the class II type, but its self-sufficiency depends on fusion of the P450 and the reductase domains on one gene and is thus produced as a single polypeptide [14] It catalyzes the subterminal (x-1
x-3) hydroxylation of fatty acids [13,15] P450foxy closely resembles in some aspects, P450BM3 (CYP102) from Bacillus megaterium The identity of the predicted amino-acid sequence of P450foxy is closest to that

of P450BM3 in both the P450 and reductase domains We therefore concluded that P450foxy is the eukaryotic coun-terpart of P450BM3 This conclusion raises the evolutio-nary question of why phylogenetically distant organisms, such as eukaryotes (F oxysporum) and prokaryotes (B megaterium), share such closely related P450s The only obvious difference between P450foxy and P450BM3 iden-tified so far is that of their intracellular localization P450foxy is exclusively recovered in the membrane fraction

Correspondence to H Shoun, Department of Biotechnology,

Graduate School of Agricultural and Life Sciences, The University of

Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Fax: + 81 35841 5148,

Tel.: + 81 35841 5148, E-mail: ahshoun@mail.ecc.u-tokyo.ac.jp

Abbreviations: GC, gas chromatography; GC-EIMS, gas

chromato-graphy-electron impact mass chromatography; P450, cytochrome

P450, rP450foxy, recombinant P450foxy.

(Received 19 November 2001, revised 22 February 2002, accepted 25

February 2002)

like other eukaryotic P450s [14], whereas P450BM3 is a

soluble protein like other bacterial P450s

Several P450 functions are of interest with respect to

potential industrial applications [16–18] and for basic studies

The most inconvenient properties of the P450 system when

considering industrial applications would be the complexity

of its electron transport In vitro P450 function can only be

exhibited in a reconstituted mixture of many components,

yet the activity is usually very lowunder such conditions One

way to overcome this obstacle would be to use a fused

protein consisting of P450 and its reductase Okawa et al

originally constructed an artificial fused protein using the

yeast expression system and applied the system to the

bioconversion of fine chemicals [19] Thereafter, P450BM3

[20] and P450foxy [13,14] were identified as naturally fused

proteins The catalytic turnover of both P450s is

exception-ally high, possibly because they are catalyticexception-ally

self-sufficient due to the fusion of two domains The naturally

fused protein would be more useful for such applications and

attempts have made using P450BM3 [21,22]

We have produced recombinant P450foxy (rP450foxy) in

the host-vector system of Saccharomyces cerevisiae [14]

However, a more efficient production system is required for

advancing both basic and application studies of P450foxy

Here, we describe the expression of P450foxy cDNA in

Escherichia coli, which resulted in the large-scale production

of rP450foxy We also characterized the substrate specificity

and other catalytic properties

M A T E R I A L S A N D M E T H O D S

Strain, culture and media

Plasmids were constructed and rP450foxy was produced

using E coli strains JM109 and DH5a, respectively E coli

strains were cultivated in Luria–Bertani broth (1%

tryp-tone, 0.5% yeast extract, 0.5% NaCl) and Terrific Broth

(1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 0.23%

KH2PO4, 0.125% K2HPO4) containing 50 lgÆmL)1

amp-icillin (LBA and TBA, respectively)

Construction of the expression plasmid

Plasmid pCWfoxy was constructed to produce recombinant

P450foxy in E coli as follows The cDNA of P450foxy [14]

was prepared by PCR using the respective 5¢ and 3¢ PCR

oligonucleotide primers:

5¢-CATATGGCTGAATCTGTTCCGATTCCGGAAC

CGCCGGGTTATCCGCTT-3¢ and 5¢-TGTTTGCTTG

ATCTCCAAAGCGTAGTT-3¢ (mutated residues are

underlined), which have homology to the nucleotide

sequences of the 5¢ and the 3¢ ends of the P450foxy

cDNA, respectively [14] PCR products were purified,

digested by NdeI and BamHI, then ligated to the plasmid

vector pCWori+ [23] that had been digested with the same

restriction enzymes The resulting plasmid was designated

pCWfoxy Standard DNA techniques proceeded according

to Sambrook et al [24]

Preparation of recombinant P450foxy

E coli DH5a was transformed with pCWfoxy, cultured

in LBA overnight, transferred to 2 L of TBA in a 5-L

volume flask, and rotated at 120 r.p.m

When D600 ¼ 0.5, 1 mM isopropyl thio-b-D-galactoside, 0.5 mM 5-aminolevulinic acid and 1 lgÆmL)1 chloram-phenicol (final concentration) were added to the medium and the flask was further incubated for 48 h under the same conditions The cells were then harvested by centrifugation, suspended in 50 mL of buffer A (50 mM

Mops/KOH, 10% glycerol, 1.0 mMdithiothreitol, 0.1 mM

EDTA, pH 7.4) and disrupted using a French Pressure Cell Press (Sim-Aminco, NewYork, USA)

The homogenate was centrifuged at 1800 g for 15 min to remove cellular debris and unbroken cells The resulting cell free extract was centrifuged at 100 000 g for 60 min to separate the supernatant (soluble) and pellet (membrane) fractions The soluble fraction was applied to a DEAE-cellulose column (Whatman DE52) equilibrated with buffer A The column was washed, then proteins were eluted with a 0–0.3M KCl gradient in buffer A The fraction containing heme was collected, dialyzed against buffer A, and applied to a 2¢-,5¢-ADP Sepharose column (Amersham Pharmacia Biotech) equilibrated with the same buffer A dark brown fraction that was eluted with

3 mM NADPH in the same buffer was directly passed through a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with buffer A The sam-ple after this elution step was used as purified rP450foxy Spectroscopy

Optical and fluorescence spectra were measured using a Beckman DU 7500 spectrophotometer and a Hitachi F-3010 fluorescence spectrophotometer, respectively Heme was identified and determined by the pyridine ferrohemo-chromogen method using the molar absorption coefficient (e) of the chromogen of the protoheme as 34.4 mM )1Æcm)1

at 557 nm [25] FAD and FMN were identified and quantified as described by Faeder & Siegel [26] using e at

450 nm ¼ 11.5 mM )1Æcm)1 The P450 content was deter-mined using an extinction coefficient of 91 mM )1Æcm)1for the difference in the carbon monoxide (CO) difference spectrum between 450 nm and 490 nm [13] The ratio in the high/lowspin states in the bound heme of P450 was calculated as reported previously [27]

Enzyme assays Fatty acid hydroxylase was assayed as described previously [13] The reaction mixture contained 50 mMMes (pH 6.5),

1 lM FAD, 1 lM FMN, 10% glycerol, 125 lM NADPH, and 125 lM fatty acid (final concentration) The reaction was initiated by adding rP450foxy, then the A340 was followed at 30C using a Beckman DU-7500 spectropho-tometer NADPH-cytochrome c reductase was assayed as described previously [13] in the same buffer containing

125 lM NADPH and 50 lM horse heart cytochrome c (Sigma) Protein conxentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc.,

CA, USA)

Data analysis Steady state kinetic analyses for P450foxy were examined with varying concentrations of fatty acid and a saturating

concentration of the second substrate (NADPH, 125 lM).

Substrate inhibition was observed for longer fatty acid

substrates Assuming that the inhibition depended on the

second binding of fatty acid substrate at its higher

concen-trations, the data were fitted to Eqn (1) using ORIGIN

Software, in which v, e, and [S] represent the experimentally

determined initial velocity, and the enzyme and the substrate

(fatty acid) concentrations, respectively Ks represents the

dissociation constant for the second binding of S

v=e¼ kcat½S=fKmþ ½Sð1 þ ½S=KSÞg ð1Þ

Determination of the reaction products

The structures of the reaction products (hydroxyl fatty

acids) of rP450foxy were determined fundamentally as

described previously [15] by gas chromatography (GC) and

GC-electron impact-mass spectrometry (GC-EIMS) Prior

to analysis, the carboxyl group of the products was blocked

by methylation with diazomethane and the hydroxyl group

was trimethyl-silylated (TMSlated) with TMSI-H (GL

Science Ltd, Tokyo, Japan)

R E S U L T S

Production of recombinant P450foxy

A cDNA of P450foxy was expressed in E coli DH5a cells

under the control of the lac promoter resident in the

pCWori+ vector To improve production, the nucleotide

sequence on the 5¢ end was mutated without changing the

encoding amino-acid sequence so as to become more A/T

rich, with the exception of proline codons that were mutated

to CCG, the most frequently used proline codon in E coli

genes The expression and production of the recombinant

protein was confirmed by the presence of the characteristic

chromophore in the cell free extract of the transformants,

which gave a specific content of 0.021 nmol P450 Æ(mg

protein))1and a yield of 10 mg P450Æ(g wet cells))1 This

yield was much higher than that attained using the yeast

system [0.2 mg P450Æ(g wet cells))1] [14] Most P450

(>88%) was recovered in the soluble fraction like the

recombinant protein produced by the yeast system [14] but

in contrast to native P450foxy, which is cofractionated with

the membrane fraction of F oxysporum [13] The

produc-tion of rP450foxy in the E coli system was further

confirmed by Western blotting that gave a specific signal

with the predicted Mr of 118 000 (data not shown) and

dodecanoic acid-dependent NADPH oxidase activity None

of these properties characteristic of P450foxy were detected

in the extract of E coli cells that harbored only the vector

The fraction containing P450 was purified to homogeneity

from the soluble fraction at a yield of 26% The Mr

estimated by SDS/PAGE and gel filtration

132 000, respectively, indicating that rP450foxy exists as a

monomer-like native protein and rP450foxy produced by

yeast [14]

Spectral properties

The absorption spectra of purified rP450foxy (Fig 1) in

its resting oxidized, dithionite-reduced ferrous, and

car-bon monoxide (CO)-ligated forms are identical to the

corresponding spectra of native P450foxy purified from

F oxysporum [13] The CO-difference spectrum with a peak at 448 nm and a trough at 407 nm (Fig 1, inset) was also identical The calculated heme content was 0.4 mol of protoheme per mol of protein In contrast to these spectral characteristics due to the bound heme, the absorbance around 450 nm (characteristic of flavin) was not prominent, with resting rP450foxy similar to the native protein, possibly because of a lowflavin content However, the presence of flavin in rP450foxy was confirmed by the characteristic fluorescence that accom-panied the emission (excited at 450 nm) peak at 528 nm The calculated specific contents of FAD and FMN were 0.15 and 0.65 molÆ(mol protein))1, respectively Native P450foxy also has a lowcontent of these cofactors (heme and flavins) [13] These results indicate that rP450foxy is correctly folded in heterologous bacterial cells

Catalytic activities Because of the lowflavin content, the activity of purified rP450foxy was low A prior incubation with free FAD and FMN remarkably accelerated the specific activity of rP450foxy as observed with the native enzyme [28] Dodec-anoic acid hydroxylase and NADPH-cytochrome c reduc-tase were assayed for rP450foxy and compared with the findings obtained using the native [13] and recombinant protein produced in the yeast host [14] (Table 1) We found that the NADPH-cytochrome c reductase activities of P450foxy are enhanced in the presence of the substrate (fatty acid) to be hydroxylated [13] We also found the same phenomenon with rP450foxy (dodecanoic acid) (Table 1) The specific activities of rP450foxy with respect to fatty acid hydroxylase and cytochrome c reductase were similar to those for the recombinant protein produced in the yeast or the native fungal protein [13,14] These results demonstrated that rP450foxy produced by E coli is kinetically and spectrally (above results) indistinguishable from the native protein

Fig 1 Absorption spectra of rP450foxy Solid line, resting (oxidized); dotted line, dithionite-reduced; dashed line, dithionite-reduced + CO Inset, CO-difference spectrum (CO-bound minus dithionite-reduced); 7.2 l M purified rP450foxy in 100 m M sodium phosphate buffer (pH 7.3), 10% glycerol, at room temperature.

We determined the substrate specificity of rP450foxy

against saturated fatty acids Figure 2 shows that rP450foxy

was active against fatty acids with a chain length of C9 (nine

carbon atoms, nonanoic acid) to C18 (18 carbon atoms,

octadecanoic acid), with the activity on tridecanoic acid

(C13) being the highest These results are similar to those

obtained using the native protein [13], but rP450foxy can

also efficiently use the shorter substrate, nonanoic acid (C9)

Stoichiometry between the consumption of NADPH and

O2 was 1.3 : 1 with all of the enzymatic reactions on

substrates with chains of C10 to C15 in length (data not

shown), consistent with the theoretical value for 2 electron

reduction coupling to monooxygenation of the substrates

Interaction of the resting rP450foxy with the substrate

fatty acids

Spectral changes were observed upon mixing the resting

rP450foxy with saturated fatty acids with chain length from

C8 to C18 They were typical of type I spectral change that

is generally observed upon binding to P450 of the substrate

to be hydroxylated [29] That is, the ratio in the high-spin

state heme with a Soret peak at around 388 nm increased

and the low-spin state heme with a peak at around 418 nm

decreased (Fig 3) The extent of the fatty-acid-chain length

that could afford the spectral change (C8-C18) almost

completely agreed with the substrate specificity determined

with respect to the overall activity above (C9-C18) The Kd value of the enzyme–substrate complex (Table 2) and the maximal absorbance change can be obtained by the spectrophotometric titration using this spectral change As observed in Fig 3, addition of the substrate did not cause a complete exchange from lowto high spin states The ratio of high-spin state heme was obtained for each rP450foxy– fatty-acid complex from the maximal absorbance change (Table 2) [27] The ratio of high spin was higher for the complex w ith a longer fatty acid up to C15 We could not determine this ratio with the longer fatty acids (C16-C18) because of the extremely lowwater-solubility of these fatty acids The spectral change with octanoic acid linearly increased up to a substrate concentration of 3 mM, w hich is the determination limit defined by the solubility of the fatty acid, indicating that the Kd for octanoate would be over

3 mM These results showed that the heme in rP450foxy is in equilibrium between high and low spin states when a fatty acid substrate binds and that the chain length of the substrate affects the equilibrium

Steady-state kinetics Apparent Kmand kcatvalues for fatty acid substrates were determined (Table 2) The Kmvalue was in a similar range (8–36 lM) between the substrates from C12 to C16, and each value approximately agreed with the respective Kd value for the same substrate In contrast, the Km value increased with decreasing chain length of the substrate

Table 1 Catalytic activities of native and recombinant P450foxy Data are mean values of three experiments.

Enzyme

Activity (nmol NADPHÆmin)1Ænmol P450)1) Dodecanoic acid

hydroxylase

Cytochrome c reductase

Cytochrome c reductase (with 150 l M dodecanoic acid)

a

Kitazume et al [14].bNakayama et al [13].

Fig 2 Apparent substrate specificity of rP450foxy for saturated fatty

acids Enzymatic activity was assayed at a fixed concentration (125 l M )

of fatty acid (C9-C18) C means control reaction without fatty acid.

Data are mean values of three experiments.

Fig 3 Spectral perturbation in rP450foxy caused by pentadecanoic acid (A) Absorption spectra of rP450foxy [6 l M in 50 m M Mes (pH 6.5), 1 l M FAD, 1 l M FMN, 10% glycerol, at 30 C] in the presence of 0, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50 l M pentadecanoic acid, respectively (lines 1–10) (B) Difference spectra Each difference spec-trum was obtained by subtracting line 1 from each of lines 2–10 in A.

belowC12 (dodecanoic acid), and became larger than the

Kdvalue for the same substrate The kinetic constants for

fatty acids over C16 could not be obtained because of the

substrate inhibition The kcat value was of the same

magnitude for all substrates tested (1200–1800 min)1)

Substrate inhibition occurred for fatty acids with a chain

length of C13 or longer (Fig 4) The inhibition was

apparent at higher substrate concentration and was

eluci-dated by the mechanism described in Eqn (1), in which a

second substrate binds to form an abortive

enzyme-substrate complex Such inhibition was not evident when

the chain length of the fatty acids was shorter than C13 (C9

to C12) The data fit closely with the respective curves

calculated on the basis of Eqn (1) with the Ks for

tetradecanoic, pentadecanoic and hexadecanoic acids being

1100, 770, and 70 lM, respectively (Table 2) These results

indicate that the substrate inhibition is stronger for longer

fatty acid substrates The substrate inhibition can elucidate

well the apparent discrepancy that the apparent activity

decreased for substrates longer than C13 (Fig 2) although

the catalytic efficiency (kcat/Km) paradoxically increased

(Table 2) Such substrate inhibition with P450BM3 has not

been reported

We also conducted assays to determine the kinetic

constants for the electron donors, NADPH and NADH,

using a saturated concentration (150 lM) of dodecanoic

acid as the substrate The Kmfor NADPH was too low to be determined, and may have been in the order of 10)6Mor lower The Kmfor NADH was 74 lM These results are same as those obtained with the native enzyme [13] Determination of the reaction products

We identified the metabolites (reaction products) of fatty acids due to the enzymatic reaction of rP450foxy by GC-EIMS The results using dodecanoic acid (C12) are shown in Fig 5 The derivatives of products were separated into three peaks on GC (Fig 5A), each of which gave a fragment pattern typical of TMSlated alcohol on electron impact mass chromatography (EIMS)

each fragment identified these metabolites as x-1, x-2 and x-3 hydroxy derivatives of dodecanoic acid, respectively The ratio of these metabolites was estimated from the signal intensity on GC (Fig 6) These results using a C12 fatty acid are similar to those we obtained using cell-free extracts of

F oxysporum[15] However, the present study is the first to identify the reaction products of purified P450foxy We also confirmed that the time-dependent decrease of the substrate during the enzymatic reaction approximately agreed with the accompanying increase in the sum of the products (data not shown) The same sets of experiments were replicated for, decanoic and undecanoic acids as substrates, and the ratio of the three products is shown in Fig 6

D I S C U S S I O N

We produced rP450foxy using an E coli expression system

in a yield that was 50-fold higher than that obtained using the yeast system [14] The recombinant protein was produced as a soluble protein unlike native fungal P450foxy that is membrane-bound However, the known catalytic and spectral properties of rP450foxy are indistinguishable from those of the native protein, supporting our previous conclusion that native P450foxy is targeted to the mem-brane by post (or co) translational modification in fungal cells We also showed that the enzymatic reaction of P450foxy exhibits a similar regiospecificity (x-1
x-3) of the reaction products for fatty acids with shorter chains (C10, C11) to that for longer fatty acids [15] We generated far more purified P450foxy using this system than the original fungal cells [13] or the yeast host-vector system [14] Thus, this unique P450 can be more extensively studied

Table 2 Kinetic constants of rP450foxy for various saturated fatty acids Data are mean values for more than five experiments Standard errors are below20% ND, not determined.

Substrate a

K d

(l M )

K m

(l M )

K s

(l M )

k cat

(min)1)

k cat /K m

(min)1Æl M )1 )

Spin state high spin(%)

a Kinetic constants could not be determined using octanoic, heptadecanoic and octadecanoic acids as substrates.

Fig 4 Steady state kinetics of long-chain fatty acids Substrates are

tridecanoic acid (j), tetradecanoic acid (d), pentadecanoic acid (s),

and hexadecanoic acid (h).

Analyses by steady state kinetics revealed a novel feature

of P450foxy with respect to substrate specificity We

examined substrates shorter than C10 (C8, C9), and found

that the C9 fatty acid can be a significant substrate although

the Km value was very large (Table 2) The present and

previous results [13] showed that P450foxy has activity for

C9-C18 fatty acids, although fatty acids longer than C18

have not been examined Regardless, the substrate

specif-icity of P450foxy is distributed among saturated fatty acids

with a rather shorter chain length than those for P450BM3

(C12-C20) The apparent activity at a fixed substrate

concentration was highest against a C13 fatty acid (Fig 2)

whereas the catalytic efficiency (kcat/Km) was higher against

longer substrates (Table 2) This discrepancy can be

explained by the inhibition that increased with longer

substrates Both Kmand Kdvalues were similar for longer

fatty acids whereas Km became much larger than Kdfor

fatty acids shorter than C12 As Km ¼ (kcat+ koff)/kon

and K ¼ k /k , w here k and k are, respectively, the

rate constants for association and dissociation of the substrate, and kcat is almost constant for all substrates tested (Table 2), the results mean that both kon and koff values became much smaller when the chain length of the fatty acid decreased In other words, rapid equilibrium cannot be assumed for the enzymatic reaction with short chain substrates

Here, we discovered a prominent difference in the catalytic properties of P450foxy and P450BM3, namely, substrate inhibition with P450foxy This could be explained

by the binding of the second substrate (Eqn 1) At present, the structural basis of the substrate-binding site of P450foxy

is not known, but alignment of the amino-acid sequences between P450foxy and P450BM3 implies similarities with the interaction of P450foxy with fatty acid substrates Crystallographic and mutational studies have shown that several amino-acid residues are critical for the binding of substrate to P450BM3 Arg47 and Tyr51 are located at the entrance of the substrate-accessing channel, and their guanidinium and hydroxyl groups, respectively, play crucial roles in the binding of the substrate by interacting with the carboxyl group of the substrate fatty acids [30–32] Phe42 is located close to the entrance and its aromatic residue may be

a lid that excludes solvent water to strengthen the electro-static interaction of Arg47 and the substrates [30–32] The corresponding amino-acid residues to those in P450foxy are Leu43, Lys48, and Phe52, respectively [14] Inside the entrance, the hydrophobic residues, Leu75, Phe87, Leu181, Ile263, and Leu437, form a hydrophobic stretch in P450BM3 that allowaccess to the aliphatic chains of fatty acids All of these residues are conserved in P450foxy These alignments indicate that all of the key amino-acid residues at the entrance of the active site pocket of P450BM3 (Phe42, Arg47, and Tyr51) are replaced by others (Leu43, Lys48, and Phe52) in P450foxy although all

of other key residues inside the pocket are conserved The positive charge essential for fixing the carboxylate of fatty acids is maintained by replacing Arg with Lys However, hydrophobicity at the entrance to P450foxy should be significantly increased as the result of the substitutions from Phe to Leu and from Tyr to Phe Why substrate inhibition is observed only with P450foxy may be explained by these substitutions The first interaction of P450BM3 with fatty acids may occur between the carboxylate anion of the

Fig 5 Gas chromatographic separation (A) and EIMS-spectra of TMSlated and methyla-ted derivatives of the reaction products (B–D) fromdodecanoic acid Mass spectra of deriva-tives 1–3 (A) are shown in B–D, respectively Relative abundance of ions in panel D was expanded by threefold for fragments with high m/z-values (indicated by horizontal arrow).

Fig 6 Regio-specificity of reaction products determined with the

substrates, decanoic (C10), undecanoic (C11), and dodecanoic (C12)

acids Relative amount of each product was determined by the extent

to which corresponding peaks separated on GC Closed, open,

and striped bars represent x-1, x-2, and x-3 hydroxy fatty acids,

respectively.

substrates and Arg47 and Tyr51 residues of the protein [30–

32] The aliphatic head of fatty acids would then turn and

penetrate the access channel The more hydrophobic

environment around the entrance of P450foxy would permit

another fatty acid molecule to partially penetrate the

channel from its aliphatic head even after the first molecule

has already occupied the channel The stronger substrate

inhibition by longer fatty acids suggests a larger

contribu-tion of the aliphatic chain of fatty acids to their binding, as

compared with P450BM3 In other words, the interaction of

P450foxy with the carboxylate anion of fatty acids at the

entrance is relatively weaker than that of P450BM3 This

notion is supported by the fact that Phe52 in P450foxy

replaces Tyr51 in P450BM3, which makes it impossible to

have the hydrogen bonding that supports the electrostatic

interaction of the positive charge (Lys48 in case of

P450foxy) with the carboxylate The effectiveness of shorter

fatty acids as substrates for P450foxy also agrees with this

replacement, as the interaction at the entrance must be more

flexible for the aliphatic head with a shorter chain to reach

the active site near heme The fact that such important

amino-acid residues are not conserved between P450foxy

and P450BM3 is notable These results demonstrate that

P450foxy and P450BM3 would be an interesting basis for

protein engineering studies from both basic and application

aspects

A C K N O W L E D G E M E N T S

This study was supported by PROBRAIN (Program for Promotion of

Basic Research Activities for Innovative Biosciences), SBPB (Structural

Biology Sakabe Project) of FAIS (Foundation for Advancement of

International Science), and Grant-in-Aid for Scientific Research from

Ministry of Education, Science, Culture and Sports of Japan.

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