(Received for publication, September 7, 1995)
From the
The isopenicillin N synthase of Cephalosporium acremonium (cIPNS) involves a catalytically important non-heme iron which is
coordinated credibly to histidine residues. A comparison of the IPNS
genes from various microbial sources indicated that there are seven
conserved histidine residues. These were individually replaced by
leucine residues through site-directed mutagenesis, and the sites of
mutation were confirmed by DNA sequencing. The seven mutant genes were
cloned separately into the vector pET24d for expression in Escherichia coli BL21(DE3), and the proteins were expressed as
soluble enzymes. All the resulting mutant enzymes obtained have
mobilities of 38 kDa, identical with the wild-type enzyme on
SDS-polyacrylamide gel electrophoresis, and were also reactive to cIPNS
antibodies. The enzymes were purified by ammonium sulfate precipitation
and DEAE-Sephadex A-50 ion exchange chromatography, and these were
analyzed for enzyme activity. A group of mutant enzymes, H49L, H64L,
H116L, H126L, and H137L, were found to be enzymatically active with
reduced activities of 16-93.7%, indicating that they are not
essential for catalysis. Two of the mutant enzymes, H216L and H272L,
were found to have lost their enzymatic activity completely, indicating
that both His-216 and His-272 are crucial for catalysis. It is
suggested that these histidines are likely to serve as ligands for
binding to the non-heme iron in the IPNS active site. Alignment of the
amino acid sequence of IPNS to related non-heme
Fe
-requiring enzymes indicated that the two essential
histidine residues correspond to two invariant residues located in
highly homologous regions. The conservation of the two closely located
histidine residues indicates the possible conservation of similar
iron-binding sites in these enzymes.
In the biosynthesis of the -lactam antibiotics, penicillin
and cephalosporin, one of the key steps is the formation of the
-lactam ring. The enzyme responsible for this reaction is
isopenicillin N synthase (IPNS) (
)which catalyzes the
oxidative cyclization of
-L-(
-aminoadipyl)-L-cysteinyl-D-valine
(ACV) into the first
-lactam intermediate, isopenicillin N. In
this reaction, four hydrogen atoms are removed with the subsequent
reduction of one molecule of oxygen into water(1) . For minimal
catalytic activity, the IPNS reaction requires the presence of iron and
ascorbate, which indicates that ferrous ions are essential for its
enzymatic activity(2) . Indeed, results from electron
paramagnetic resonance (EPR), Mössbauer, and
electronic absorption spectroscopic analyses of the IPNS from Cephalosporium acremonium (cIPNS) have revealed that the
active site contains ferrous ions(3) . In addition,
stoichiometry of one molecule of Fe
per molecule of
the enzyme has been determined for the cIPNS enzyme(3) .
Spectroscopic analysis by nuclear magnetic resonance (H
NMR) studies have implied that the Fe
in the native
IPNS is six-coordinated with nitrogen- and/or oxygen-containing
ligands, of which three could correspond to imidazole
moieties(4) . The possible involvement of histidine residues as
imidazole ligands in the active site of the enzyme was thus suggested.
Supporting the role of histidine residues in coordinating
Fe
, studies of cIPNS involving multiple scattering
analysis of the extended x-ray absorption fine structure data indicated
that two or three histidines are ligated to the
Fe
(5) . In separate studies, electron spin
echo envelope modulation analysis of the
Cu
-substituted cIPNS revealed two equatorially
coordinated histidines in addition to a water molecule(6) .
The IPNS enzyme has been found in a number of prokaryotes and lower eukaryotes known to produce penicillin and cephalosporin. The pcbC gene coding for the IPNS enzyme has been cloned from C. acremonium(7) , Aspergillus nidulans(8) , Penicillium chrysogenum(9) , Streptomyces clavuligerus(10) , S. lipmanii(11) , S. jumonjinensis(11) , S. griseus(12) , Nocardia lactamdurans(13) , and Flavobacterium(14) . Comparison of the amino acid sequences of these enzymes revealed that they share high homology of 57.1% to 83.1%(15) . It has thus been difficult to define amino acids or regions of functional importance. However, since spectroscopic studies have implicated histidine residues as candidates for iron ligands, the conservation of histidine residues could be examined by sequence alignment of all nine known IPNS proteins. In such an analysis, it was observed that, of the ten histidine residues in cIPNS, seven at positions 49, 64, 116, 126, 137, 216, and 272 (with cIPNS sequence as reference) are conserved as illustrated in Fig. 1. We have recently shown that His-272 is essential for the catalytic activity of cIPNS, possibly as a ligand for the non-heme iron(16) . In the present study, we used site-directed mutagenesis to assess the importance of each of the other conserved histidine residues for the enzyme activity of recombinant cIPNS expressed in Escherichia coli.
Figure 1: Alignment of the amino acid sequences of isopenicillin N synthases. The conserved histidine residues are shaded. Amino acid residues are numbered from the first base of the ATG initiator codon.
Figure 2: A, SDS-PAGE analysis of cell-free extracts derived from E. coli BL21(DE3) transformants. Lane 1 contains the markers used with the molecular masses indicated on the left. The following lanes were each loaded with 5 µg of cell-free extract from E. coli BL21(DE3) transformant containing the following vector: in lane 2, pET24d; lane 3, pET24d/wild-type cIPNS; lane 4, pET24d/H49L-cIPNS; lane 5, pET24d/H64L-cIPNS; lane 6, pET24d/H116L-cIPNS; lane 7, pET24d/H126L-cIPNS; lane 8, pET24d/H137L-cIPNS; lane 9, pET24d/H216L-cIPNS; lane 10, pET24d/H272L-cIPNS. The gel was stained with Coomassie Brilliant Blue dye. B, immunoblotting analysis of an exact replica of the SDS-PAGE gel. The proteins were transferred onto nitrocellulose membrane, and this was probed with serum containing antibodies raised against wild-type cIPNS. The arrowheads indicate the position of cIPNS. In the control lane (lane 2), no cross-reaction was detected.
All seven conserved histidine residues in cIPNS were modified using site-directed mutagenesis by substituting each of these histidine residues with leucine. After establishing the site of mutation by DNA sequencing (data not shown), the mutated genes were individually cloned into the expression vector pET24d in the same manner as the wild-type cIPNS gene and were expressed under similar conditions. SDS-PAGE analysis revealed that the wild-type and the various mutant cIPNS proteins exhibited identical mobilities (Fig. 2A). The proteins also possessed similar immunoreactivity toward the antibodies specific to cIPNS (Fig. 2B), despite differences in the level of expression.
The enzyme activities of the wild-type cIPNS and its mutant enzymes were assayed by HPLC analysis method using cell-free extracts harboring the respective enzymes. As compared to the wild-type enzyme, these mutant enzymes, H49L, H64L, H116L, H126L, and H137L, were found to be enzymatically active but less so than the wild-type enzyme. The relative specific activities of these mutant enzymes ranged from 8.5% to 86.6% as determined by HPLC assay (Table 2). The enzyme assays were carried out in duplicate measurements on at least three dilutions of lysates containing the respective mutant proteins and repeated at least four times with substantially the same results in each case. The results obtained indicated that these five histidine residues do not play essential roles in catalysis, although substitution with leucine does influence the level of active enzyme recovered, possibly by affecting enzyme expression, stability, specific activity, or substrate affinity, or a combination of these factors.
No IPNS enzyme activity was detected for the H216L and H272L mutant enzymes in all the determinations by HPLC analysis (Table 2). When the same samples were separated on SDS-PAGE and subjected to immunoreactivity to IPNS antisera on Western blots, the amounts of H216L and H272L mutant enzymes detected were found to be comparable with that of the wild-type cIPNS (Fig. 2).
Figure 3: SDS-PAGE analysis of the purified wild-type and mutant cIPNS enzymes. The proteins were purified through ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography. The molecular mass markers (in kDa) are indicated on the left. Lane 1 shows the cell-free extract from E. coli expressing the wild-type cIPNS, and lane 2 shows the 55-85% ammonium sulfate-saturated fraction of the cell-free extract. The following lanes were each loaded with the purified proteins as indicated: in lane 3, wild-type cIPNS; lane 4, H49L-cIPNS; lane 5, H64L-cIPNS; lane 6, H116L-cIPNS; lane 7, H126L-cIPNS; lane 8, H137L-cIPNS; lane 9, H216L-cIPNS; lane 10, H272L-cIPNS. The gel was stained with Coomassie Brilliant Blue dye.
Enzyme activity determination of the purified wild-type and mutant enzymes by the bioassay method confirmed that alteration of His-49, His-64, His-116, His-126, and His-137 in cIPNS resulted in the reduction of enzymatic activities (Table 3). The activities of these purified mutant enzymes ranged from 16% to 93.7% relative to that of the wild-type enzyme, while the purified mutant H216L and H272L enzymes did not reveal any catalytic activity. Thus, substitution of His-216 and His-272 by leucine resulted in the production of immunoreactive mutant cIPNS that failed to give detectable activity.
Since the IPNS gene was first cloned from C. acremonium(7) , there has been much interest in determining functionally important amino acids involved in the catalytic function of IPNS. Thus, much information has been accrued from studies of the primary structure of IPNS(7, 8, 9, 10, 11, 12, 13, 14) , spectroscopic analysis of the enzyme(3, 4, 5, 6, 26, 27, 28) , affinity labeling of the substrate binding sites(29) , chemical modification of reactive groups(2) , and site-directed mutagenesis studies(16, 30) . Based on chemical modification studies(2) , cysteine residues were thought to play an important role in the IPNS catalytic center. However, site-directed mutagenesis of two cysteine residues, Cys-106 and Cys-255 of cIPNS, followed by enzyme activity studies revealed that they were not essential for IPNS activity(30) . This has led us to investigate the functional role of histidine residues in cIPNS through site-directed mutagenesis of conserved histidine sites since spectroscopic studies have indicated the involvement of histidines as iron ligands(3, 4, 5, 6, 26, 27, 28, 29) . Thus, in our previous analysis, a catalytically important residue His-272 of cIPNS was proposed as a possible ligand for iron binding(16) . In this study, evaluation of the activity of the same H272L mutant enzyme by HPLC determination and analysis of the purified enzyme confirmed further that this mutant enzyme lacks any detectable activity, thereby substantiating the importance of His-272 for IPNS catalysis.
The search for conserved histidine residues that
might be functional in enzyme catalysis has been investigated by amino
acid sequence comparisons of a number of related non-heme
Fe-requiring enzymes including the IPNS enzymes (Fig. 4). These studies indicated that
His-49(31, 33) , His-216(32, 33) ,
and His-272 (31, 32, 33) of cIPNS are
conserved in regions of relatively high sequence homology. Among the
enzymes compared are 2-oxoglutarate-dependent dioxygenases (prolyl and
lysyl hydroxylase) from animal primary metabolism,
2-oxoglutarate-dependent hydroxylases (hyoscyamine 6
-hydroxylase,
flavanone 3
-hydroxylase, deacetoxycephalosporin C synthase, and
deacetylcephalosporin C synthase) from plant, fungal, and bacterial
secondary metabolism, and enzymes involved in ethylene formation and
anthocyanidin biosynthesis in plants. In a number of these enzymes, e.g. prolyl hydroxylases and flavanone hydroxylases, the
importance of histidine residues to their catalytic mechanism has been
demonstrated through chemical modification studies (32, 33) . In addition, it was demonstrated recently
that site-directed mutagenesis of human prolyl 4-hydroxylase (34) identified three histidine residues to be critical for
activity, possibly as ligands for iron binding, and two of these
correspond to the two essential histidine residues that have been
experimentally shown to be important for the catalysis of cIPNS. One
similar feature of these enzymes including IPNS is that they require
ferrous iron and oxygen for activity. In view of the common requirement
for ferrous iron to invoke activity in all the enzymes studied, albeit
from uncommon sources, it appears that the homologous sequences in the
vicinity of certain histidine residues of these enzymes might represent
part of the iron-binding site. The conservation of the histidine
residues provides strong support for the possible conservation of
similar iron-binding sites in these enzymes.
Figure 4:
Comparison of amino acid sequences of IPNS
enzymes and related enzymes showing homologous regions with the
conserved histidine residue (*). The aligned sequences are as
indicated: 1-9, IPNS from C. acremonium, P.
chrysogenum, A. nidulans, N. lactamdurans, S. clavuligerus, S.
jumonjinensis, S. griseus, S. lipmanii, and Flavobacterium; 10, hyoscyamine 6-hydroxylase from Hyoscyamus niger; 11-16, ethylene-forming
enzymes (aminocyclopropane-1-carboxylic acid oxidase) from Persea
americana, Brassica juncea, Arabidopsis thaliana, Lycopersicon
esculentum, Dianthus caryophyllus, and Petunia hybrida; 17-22, flavanone 3-hydroxylase from P. hybrida,
Matthiola incana, Callistephus chinesis, D. caryophyllus, Antirrhinum
majus, and Hordeum vulgare; 23 and 24,
anthocyanidin synthase from Zea mays, A. majus; 25,
chick lysyl hydroxylase; 26, human prolyl hydroxylase,
-subunit; 27, deacetoxycephalosporin C synthase (DAOCS)/deacetylcephalosporin C synthase (DACS) from C. acremonium; 28, DAOCS from S.
clavuligerus; 29, DACS from S. clavuligerus.
Amino acids that are identical with those of cIPNS are shaded.
A recent analysis involved the comparison of the sequences of penicillin and cephalosporin biosynthetic enzymes, viz. nine IPNS enzymes from various microbial sources, deacetoxycephalosporin C synthase and deacetylcephalosporin C synthase from S. clavuligerus, and the C. acremonium deacetoxycephalosporin C synthase/deacetylcephalosporin C synthase (35) . From the alignment of the primary protein structure of these enzymes and a comparison of their putative secondary structures, two histidine residues equivalent to His-216 and His-272 of cIPNS were found to be aligned consistently in the 12 proteins compared. These two histidine residues were thereupon predicted to have the highest probability of being iron-binding ligands.
Our experimental investigations are in
support of this predictive analysis and have also identified His-216
and His-272 in cIPNS as being necessary for the catalytic activity of
cIPNS. The essential function of both His-216 and His-272 in cIPNS was
implicated by the loss of IPNS activity upon replacement of the
histidine residues individually with leucine. The removal of the
imidazole side chain of these histidine residues with the concomitant
loss of activity indicated that these side groups are involved in
catalysis. Although His-49 of cIPNS was also found to be conserved in a
relative position in a number of the non-heme
Fe-requiring enzymes, the corresponding site-specific
mutant enzyme was found to retain some enzyme activity indicating that
a histidine moiety at this position is not crucial for cIPNS activity.
Similar replacement of the other four conserved histidine residues with
leucine also did not totally abolish enzyme activities in the
site-specific mutant enzymes, suggesting that they are not important
for IPNS catalysis.
Previous spectroscopic studies have postulated
binding of a third histidine to the metal center of the
FeIPNS holoenzyme and the
Fe
IPNS
ACV complex, but which is
subsequently displaced by solvent or oxygen
binding(4, 26) . However, the results of the
site-directed mutagenesis analyses indicate that only two histidine
residues are definitely required for the activity of IPNS. In the
recently published crystal structure of Mn
IPNS
from A. nidulans, two histidines were also indicated in the
metal ion active site. The proposed three-dimensional structure of the
holoenzyme reveals that a manganese ion is attached to four protein
ligands (His-214, Asp-216, His-270, and Gln-330 of A. nidulans IPNS) and two water molecules. The two histidine ligands
identified corresponded to His-216 and His-272 of C. acremonium IPNS.
Current studies have indicated that in the complex
FeIPNS
ACV
NO, at least three iron
coordination sites can be occupied simultaneously by exogenous ligands,
namely, ACV, water, and
NO(3, 4, 5, 27, 28) . In
line with this, it was proposed that ACV and oxygen bind to the
coordination sites occupied by the water molecules and the C-terminal
glutamine(36) . This implies that the ferrous ion at the active
site could be bound to three endogenous ligands, Asp-218, His-216, and
His-272 (of cIPNS) throughout the catalytic cycle. Our results are in
support of this arrangement in that the only two histidine residues
corresponding to His-216 and His-272 in cIPNS are shown to be essential
for IPNS activity.
The role of paired histidines as putative
non-heme iron ligands has also been defined by site-directed
mutagenesis of human lipoxygenase (37) whereby a non-heme iron
has been demonstrated to be important for enzyme activity. In the rat
liver phenylalanine hydroxylase which requires a non-heme
Fe for the conversion of phenylalanine to tyrosine,
two histidine residues have also been proposed to bind the
iron(38) . Further studies on the iron content or iron affinity
in the site-specific mutant proteins is warranted for elucidating a
clearer role for the paired histidines in IPNS.