AbstractThreonine-301 from rabbit liver cytochromes P-450 (laurate (ω-1)-hydroxylase and testosterone 16α-hydroxylase) has been replaced by histidine via site-directed mutagenesis. In the oxidized state the mutant P-450s exhibited typical low-spin type absorption spectra of P-450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral change was induced on addition of substrates for their wild-type counterparts. The mutant P-450s were also completely devoid of the hydroxylase activity. These findings suggest that threonine-301, which is highly conserved in P-450s and located at the distal heme surface, plays an important role in substrate binding

Imai, Yoshio

Nakamura, Masahiko

Elsevier - Publisher Connector 

Volume 234, number 2, 313-315 FEB 06054 July 1988 
The importance of threonine-301 from cytochromes P-450 (laurate 
(w- 1)-hydroxylase and testosterone 1 6a-hydroxylase) in substrate 
binding as demonstrated by site-directed mutagenesis 
Yoshio Imai and Masahiko Nakamura 
Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan 
Received 8 April 1988 
Threonine-301 from rabbit liver cytochromes P-450 (laurate (w-l)-hydroxylase and testosterone lba-hydroxylase) has 
been replaced by histidine via site-directed mutagenesis. In the oxidized state the mutant P-450s exhibited typical low-spin 
type absorption spectra of P-450 and their reduced CO complexes showed a Soret peak at 450 nm. However, no spectral 
change was induced on addition of substrates for their wild-type counterparts. The mutant P-450s were also completely 
devoid of the hydroxylase activity. These findings suggest that threonine-301, which is highly conserved in P-450s and 
located at the distal heme surface, plays an important role in substrate binding. 
Cytochrome P-450; Laurate; Testosterone; Hydroxylation; Site-directed mutagenesis 
1. INTRODUCTION 
Cytochromes P-450 constitute a large family of 
heme-thiolate proteins involved in oxidative 
metabolism of a wide variety of xeno- and en- 
dobiotic compounds. In the ferric low-spin state 
the heme iron of P-450 is hexacoordinated and a 
water molecule or hydroxide anion occupies the 
axial 6th coordination position trans to the thiolate 
5th ligand [l]. X-ray crystallographic studies have 
shown that threonine-252 of P-450cam from 
Pseudomonas putida is located at the distal heme 
surface [l] and this threonine residue is highly con- 
served in P-450s [l-3]. On the other hand, the 6th 
coordination position is thought to be occupied by 
a histidine imidazole in P-450~~1, an altered P-450 
from a yeast mutant defective in lanosterol 14a- 
Correspondence address: Y. Imai, Institute for Protein 
Research, Osaka University, Suita, Osaka 565, Japan 
Abbreviations: P-450, cytochrome P-450; P45O(w-l), P-450 
(laurate (w-1)-hydroxylase); P-450(1&), P-450 (testosterone 
16cu-hydroxylase); P-450~~1, P-450 from a yeast mutant defec- 
tive in lanosterol 14cu-demethylation; P-45&am, Pseudomonas 
putida P-450 
demethylation [4]. We have recently constructed 
two plasmids for expression in yeast of rabbit liver 
P-450&-l) and P-450(16cu) and characterized the 
P-450s synthesized in yeast transformed with these 
expression plasmids [5,6]. In this study we con- 
structed mutants of P-45O(w-1) and P-45O(lti) by 
site-directed mutagenesis to examine a possible 
role of the aforementioned conserved threonine in 
catalytic activity and report our findings therein. 
2. MATERIALS AND METHODS 
Restriction endonucleases were purchased from Nippon 
Gene, T4 DNA ligase (DNA ligation kits) from Takara Shuzo, 
and Ml3 cloning, sequencing, and in vitro mutagenesis kits 
from Amersham International. The mutagenic oligonucleotide 
primer, 5’-CTGGAACAGAGCACACAAGCACCA-3’) and 
the cDNA sequencing primer (for screening of the mutants), 
5 ’ -CCCTCGGGACTTTATTG-3 ’ , were prepared by the 
phosphoramidite method [7] using an automated DNA syn- 
thesizer (Applied Biosystems model 381A). pAH3P2 and 
pAHF3, expression plasmids for P-45O(w-1) and P-450(1&), 
respectively, were described in [5,6]. The yeast expression vec- 
tor pAAH [S] was supplied by Dr A. Ito of Kyushu University, 
and Saccharomyces cerevisiae strain AH22 by Dr Y. Oshima of 
Osaka University. 
Site-directed mutagenesis was performed by the method of 
Nakamaye and Eckstein [9] according to the instruction pro- 
Published by Elsevier Science Publishers B. V. (Biomedical Division) 
00145793/88/$3.50 0 1988 Federation of European Biochemical Societies 313 
Volume 234, number 2 FEBS LETTERS July 1988 
vided by the kit supplier. Expression plasmids for the mutated 
P-450s were constructed from the mutated P-450 cDNAs and 
pAAH as described [S]. Transformation and cultivation of 
yeast, preparation of yeast microsomes and purification of 
P-450s were carried out by the methods described previously 
[5,6]. Reconstituted hydroxylase activities were measured as 
described [5,6,10]. Nucleotide sequences were determined by 
the dideoxy termination method [l 11. 
3. RESULTS AND DISCUSSION 
Sequence alignment of P-450s indicates that 
threonine-252 of P-450cam corresponds to 
threonine-301 of P-450&-l) and P-450(16&) [2,3], 
and is located in a threonine-serine cluster that is 
invariably found in rabbit liver P-450s [2]. To 
replace this threonine by histidine, the inserts of 
pAH3P2 and pAHF3 (1.78 and 1.64 kbp Hind111 
fragments, respectively) were cloned into 
M13mpll and subjected to site-directed muta- 
genesis (see section 2). The desired mutants were 
selected by sequencing of the single-stranded DNA 
insert prepared from progeny phages (fig. 1). Com- 
plete sequencing of the inserts also confirmed that 
unwanted mutations had not occurred in the 
selected mutants. Each of the mutated P-450 
cDNAs thus obtained was inserted into the Hind111 
site of the expression vector pAAH5. Microsomes 
prepared from yeast transformed with the expres- 
sion plasmids having the mutated cDNAs con- 
tained spectrophotometrically detectable P-450, 
although its content was lower than in microsomes 
from yeast carrying pAH3P2 or pAHF3 (not 
shown). The mutated P-450% P45O(w-1 :T301H) 
and P_450(16a:T301H), were solubilized from the 
corresponding microsomes and purified partially 
as described [5,6] with a yield of 3-5 nmol from 
3 1 of culture. 
As shown in fig.2, both mutant P-450s in the ox- 
idized state show absorption spectra that are 
typical of a low-spin type of P-450 (Soret band at 
416-417 nm, cy band 2 ,B band) and similar to that 
of wild-type P-450(1&) [6]. Since wild-type 
P-45O(w-1) exhibits a mixed-spin type spectrum in 
the oxidized state [5], it seems that replacement of 
threonine-301 by histidine in this P-450 has caused 
a change in the heme environment. At any rate, the 
spectra of the ferric mutant P-450s are clearly dif- 
ferent from that of ferric P-450~~1 (red shifted 
Soret band, LY band < p band) [4], which is essen- 
tially identical with those of I-methylimidazole 
complexes of the ferric mutant P-450s (fig.2A). 
These results indicate that the mutant P-450s have 
not a histidine imidazole as the 6th ligand of the 
ferric low-spin heme. Spectra of the l-methyl- 
imidazole complexes of the mutant and wild-type 
P-45& are superimposable on each other. Kd 
Fig.1. Sequence analysis of P-450 cDNA in the region where desired mutation was expected. The conversion of the codon for 
threonine-301 (ACA for P-45O(w-1) and ACC for P-450(1&)) to that of histidine (CAC) is marked with asterisks. 
314 
Volume 234, number 2 FEBS LETTERS July 1988 
WAVELENGTH (nm) 
Fig.2. Absolute absorption spectra of mutant P-45& (A) 
P-450&-l :T301H); (B) P-450(16a: T30lH). The partially 
purified preparation was dissolved in 100 mM potassium 
phosphate buffer (pH 7.25) containing 20% glycerol and 
detergents (0.2% Emulgen 913 for A or 0.4% sodium cholate 
and 0.1% Emulgen 913 for B). (-) Oxidized, addition of 
1 mM sodium laurate (A) or 0.5 mM testosterone (B) caused no 
change in this spectrum; (- --) and (+. .) reduced CO complex 
recorded 2 and 8 min after the addition of sodium dithionite, 
respectively; (-----) I-methylimidazole (final concentration, 
25 mM) was added to the oxidized form (A). 
values of the mutant P-450s for 1-methylimidazole 
were determined to be approximately the same as 
those of the wild-type P-450s. The reduced CO 
complexes of the mutant P-450s show a Soret peak 
at 450 nm, as is the case for their wild-type 
Table 1 
Hydroxylase activity of partially purified P-450s 
Species Substrate Metabolite Wild-typea T30lH 
mutanta 
P-450(&J-l) caprate 9-OH C 8.2 nd 
laurate 11-OH L 7.9 nd 
P-450(1&) testosterone 16cr-OH T 3.4 nd 
progesterone l&-OH P 4.7 nd 
a Results expressed as nmol/min per nmol P-450 
The activities were measured by reconstituting the hydroxylase 
system as described previously [5,6,10]. nd, not detectable 
counterparts [5,6], together with a peak at 420 nm 
due to the denatured form. These findings support 
the view that in the mutant P-450s the native axial 
ligands of the heme iron are still maintained, 
although their reduced CO complexes were very 
unstable and converted rapidly to their denatured 
forms (fig.2B). 
Despite the evidence for preservation of native 
axial ligands, the mutant P-450s could not catalyze 
the hydroxylation of substrates that can be 
metabolized by the wild-type P-450s (table 1). 
When laurate or caprate binds to ferric P-45O(w-l), 
its spectrum is converted to the typical high-spin 
type (&, 7-9pM) [5]. The addition of the fatty 
acids (up to 1 mM at least) to ferric 
P-45O(w-1 :T301H), however, induced no spectral 
change, indicating that the mutant P-450 is defec- 
tive in substrate binding. The results described 
above suggest strongly that threonine-301 plays an 
important role in substrate binding. The possibility 
still remains that the histidine residue in the place 
of the threonine residue affects the function of the 
threonine-serine cluster in which the conserved 
threonine is located. Investigations are in progress 
to examine this possibility by replacing 
threonine-301 with other amino acids. 
Acknowledgement: We are grateful to Mr J. Miyagawa for 
determination of fatty acids, steroids, and their hydroxylated 
products. 
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The importance of threonine-301 from cytochromes P-450 (laurate (ω-1)-hydroxylase and testosterone 16α-hydroxylase) in substrate binding as demonstrated by site-directed mutagenesis 

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The importance of threonine-301 from cytochromes P-450 (laurate (ω-1)-hydroxylase and testosterone 16α-hydroxylase) in substrate binding as demonstrated by site-directed mutagenesis

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