Antonie van Leeuwenhoek 75: 3339, 1999.

1999 Kluwer Academic Publishers. Printed in the Netherlands.

33

Recent advances in the structure and function of isopenicillin N synthase

Rachel Kreisberg-Zakarin, Ilya Borovok, Michaela Yanko, Yair Aharonowitz & Gerald Cohen
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv
University, Ramat Aviv 69978, Israel. ( Author for correspondence)
Received 27 August 1998; accepted in revised form 15 October 1998

Key words: active site, crystal structure,iron binding motif, isopenicillin N synthase, mechanism of action

Abstract
Isopenicillin N synthase is a key enzyme in the biosynthesis of penicillin and cephalosporin antibiotics, catalyzing
the oxidative ring closure of -(L--aminoadipoyl)-L-cysteinyl-D-valine to form isopenicillin N. Recent advances
in our understanding of the unique chemistry of this enzyme have come through the combined application of spectroscopic, molecular genetic and crystallographic approaches and led to important new insights into the structure
and function of this enzyme. Here we review new information on the nature of the endogenous ligands that constitute the ferrous iron active site, sequence evidence for a novel structural motif involved in iron binding in this and
related non-heme iron dependent dioxygenases, crystal structure studies on the enzyme and its substrate complex
and the impact of these and site-directed mutagenesis studies for unraveling the mechanism of the isopenicillin N
synthase reaction.
Abbreviations: ACG -(L--aminoadipoyl)-L-cysteinyl-glycine; ACV -(L--aminoadipoyl)-L-cysteinyl-Dvaline; IPNS isopenicillin N synthase.
Introduction
The biosynthesis of penicillin and cephalosporin antibiotics commences with two remarkable reactions,
the first catalyzed by a peptide synthetase, the second by a mononuclear non-heme dioxygenase (Figure
1). The peptide synthetase is a 450 kDa protein
that carries out the sequential joining of three amino
acids to form the tripeptide -(L--aminoadipoyl)L-cysteinyl-D-valine (ACV), and has received much
attention as a model system for studying the linear
assembly of non-ribosomal peptide antibiotics. The
nature and organization of the individual structural
and functional domains of the ACV synthetase that
are responsible for the activation, condensation and
epimerization reactions have recently been reviewed
(Marahiel 1997). In this chapter we focus on the second step in the biosynthetic pathway, the cyclization of
ACV by the enzyme isopenicillin N synthase (IPNS)
to form the -lactam and the thiazolidine rings of
isopenicillin N. In particular, we record progress in

determining the 3D structure of IPNS, the nature of

the catalytic active-site and the mechanism of action.
Other aspects of IPNS that deal more with genetic regulation, metabolic engineering and evolution of IPNS
genes have been reviewed elsewhere (Cohen et al.
1990; Skatrud 1992; Aharonowitz et al. 1992; Cohen
& Aharonowitz 1995).
IPNS Reaction
IPNS is one of a broad class of non-heme FeII
dependent enzymes that activate molecular oxygen and catalyze important reactions in secondary
metabolism, such as the biosynthesis of penicillin and
cephalosporin antibiotics in bacteria and fungi, and the
production of flavonoids, alkaloids and hormones in
plants, as well as in numerous other processes (Feig
& Lippard 1994; Prescott 1996; Que & Ho 1996).
A unique feature of IPNS (and one other enzyme in
this group, 1-amino cyclopropane 1-carboxylic oxidase (ACCO) involved in ethylene formation) is that
it carries out the complete four-electron reduction of

34

Figure 2. Coordination environment of the FeII-IPNS-ACV-NO

complex; numbers refer to the positions of histidine and aspartic
acid residues in the Streptomyces jumonjinensis IPNS.

Structure of the IPNS FeII active site

Figure 1. Role of ACV synthetase and isopenicillin N synthase in

the biosynthesis of penicillins and cephalosporins in bacteria and
fungi.

molecular oxygen to two water molecules (White et

al. 1982). In the case of IPNS these electrons originate in the substrate ACV (Figure 1). In the 1980s,
Baldwin and his co-workers examined the mechanism
of the IPNS reaction in studies employing kinetics,
stereochemistry and a host of substrate analogs. Their
results supported a multistep mechanism in which the
formation of isopenicillin N proceeds via sequential
ring closure steps to form first the -lactam ring and
then the thiazolidine ring (Baldwin & Bradley 1990).
Within the last decade, gene cloning has resulted in the
availability of substantial amounts of highly active recombinant IPNS proteins that have greatly facilitated
further physical and biochemical studies on the structure and function of IPNS, and which are summarized
below.

Spectroscopic studies of the Cephalosporium acremonium FeII-IPNS have led to a detailed picture of the
iron atom in the active site (reviewed in Cooper 1993;
Que & Ho 1996; Schofield et al. 1997). In the absence
of the substrate ACV, the ferrous iron is attached to
two histidines and one aspartic acid and to an additional endogenous ligand that subsequently was shown
from the crystal structure of the Aspergillus nidulans
IPNS to be a glutamine (Roach et al. 1995, and see
below). Two solvent molecules occupy the remaining
sites of the six-coordinate iron ion. Because binding
of ACV in the presence of O2 results in an unstable
complex, spectroscopic studies have used NO as a
useful analog of O2 to follow the formation of the FeIIIPNS-ACV-O2 adduct. The EPR spectrum, EXAFS
analysis and other spectroscopic studies of the FeIIIPNS and the ternary complex indicate that one of the
endogenous ligands and one of the water molecules
are displaced on binding of ACV, which occurs as the
thiolate. Figure 2 depicts the proposed structure of the
catalytically active complex containing three endogenous and three exogenous ligands. Crystallographic
studies show that glutamine is the endogenous ligand
displaced on substrate binding and that the NO (O2 ) is
bound to the iron center trans to the aspartate ligand
(Roach et al. 1995, and see below).
Localization of the FeII active site endogenous
ligands
Genetic studies were used to identify the histidine and
aspartic acid ligands in the FeII active site. In one
study the primary amino acid sequences of ten fungal
and bacterial IPNSs were aligned to show that seven
histidines and five aspartic acid residues are conserved
throughout. These residues were changed one at a time

35
to alanines by site directed mutagenesis in the IPNS of
Streptomyces jumonjinensis and the recombinant proteins synthesized in Escherichia coli (Borovok et al.
1996). Biochemical analysis revealed that three of the
mutant enzymes, His212, Asp214 and His 268, had no
measurable activity, whereas the other nine had specific activities ranging from 568 percent that of the
wild type enzyme. Since CD spectroscopy of the three
fully defective enzymes and the wild type showed no
significant difference in their conformation, His212,
Asp214 and His268 are, therefore, essential for IPNS
activity, and it was proposed that they constitute three
of the four endogenous FeII active site ligands identified from the spectroscopic studies. Similar studies
carried out with the C. acremonium IPNS led to the
same conclusions (Tan et al. 1996; Loke et al. 1997).
The crystal structure of IPNS verifies these findings
(see below).
Iron binding motif
Independent support for the above assignment has
come from a comparison of the primary amino acid sequences of a large group of non-heme FeII-dependent
dioxygenases, all of which activate molecular oxygen
and carry out diverse reactions such as aliphatic hydroxylations, desaturations and cyclizations. Among
these are two additional enzymes an expandase and a
hydroxylase both of which, together with IPNS, take
part in the biosynthesis of the cephalosporin class of
-lactam antibiotics (Figure 1). Sequence analysis of
some 52 members of this class of enzymes established
that five residues alone are conserved throughout
Gly41, His212, Asp214, His268 and Arg277, numbering according to S. jumonjinensis IPNS (Borovok
et al. 1996). Note that the two histidines and aspartic
acids are precisely the ones essential for IPNS activity. A more extensive analysis (Figure 3) comprising
some 139 non-redundant non-heme FeII dependent
dioxygenases revealed that both the two histidines
and the aspartic acid and the glycine are fully conserved (Kreisberg-Zakarin 1998). This analysis was
used to define a sequence motif that is common to
all the non-heme FeII dependent dioxygenases, HisX-Asp(53-57)X-His, and is presumed to be necessary
for binding of the iron in the active site (Borovok et
al. 1996). Several other residues are highly conserved,
in particular Arg277 and Ser279 which are bound in
IPNS to the ACV valine carboxylate (Roach et al.
1997) and which possibly bind to the C-5 carboxylate
of 2-oxoglutarate in the sub-group of enzymes that re-

quire it as a cofactor. No role has so far been ascribed

to the Gly41.
Compelling evidence for the generality of a common structural motif in the active-site of non-heme
FeII enzymes comes from a comparison of the crystal
structures of five enzymes in this class tyrosine hydroxylase, dihydroxybiphenyl 1,2-dioxygenase, soybean lipoxygenase, iron superoxide dismutase and
IPNS which carry out very different reactions. Hegg
& Que (1997) pointed out that each of the structures
contains two histidines and one carboxylate ligated to
the FeII atom. Although the spacing between these
ligands is quite different in the primary amino acid
sequences of the enzymes, it is significant that in each
case the two histidines and carboxylate are arrayed
on one face of the octahedral coordination sphere.
Hegg & Que have termed this arrangement the 2-His1-carboxylate facial triad and suggested that its role is
to anchor the iron in the active site while providing
access on the opposite side of the octahedron for other
exogenous ligands such as substrates and cofactors.
The possibility that the facial triad may have a catalytic as well as a functional role is suggested from
some recent studies which demonstrate that the spatial
organization of the ligands in the IPNS triad is critical
for enzyme activity (Kreisberg-Zakarin et al. 1998).
Thus, when site-directed mutagenesis was used to exchange in S. jumonjinensis the His and Asp residues
at positions 212 and 214, respectively, the mutant enzyme was completely inactive; a similar result was
found when the Asp and His residues at positions 214
and 268 were exchanged. Furthermore, conversion of
Asp214 to a histidine to create three histidine ligands
resulted in a completely inactive enzyme. Some implications of these results for IPNS mechanism are
discussed below.
Crystal structure of IPNS
The first crystal structure of IPNS was determined
for the Aspergillus nidulans enzyme complexed with
manganese instead of iron (Roach et al. 1995). Previous attempts to obtain crystals of sufficient quality for
structure determination of the C. acremonium and the
S. jumonjinensis IPNSs were unsuccessful (Fujishima
et al. 1994; J. Remington, pers. comm.). The structure
at 2.5 shows that the active site is buried within the
hydrophobic pocket of an eight-stranded jelly roll barrel which possibly functions to isolate highly reactive
intermediates from the environment. The manganese
atom is attached to four protein ligands, His214,

36

Figure 3. The two conserved histidines and one aspartate in IPNS and in other ferrous iron dependent dioxygenases that define the iron
binding motif (see text). The abridged multiple sequence alignment shows three blocks of amino acids taken from the IPNS of S. jumonjinensis and representative members of other dioxygenases in this class. Numbering according to the IPNS of S. jumonjinensis. Enzyme
designations, sources and data bank references are as follows, in descending order: IPNS_STRJU: isopenicillin N synthase (IPNS) from S.
jumonjinensis; SW:IPNS_STRJU. HYD_STRCL: deacetylcephalosporin C synthase from S. clavuligerus; SW:CEFF_STRCL. EXP_STRCL:
deacetoxycephalosporin C synthase from S. clavuligerus; PIR2:A39204. EXP/HYD_CEPAC: expandase/hydroxylase from C. acremonium;
PIR2: A29711. ANS_MAIZE: A2 gene from Z. mays; SW:ANS_MAIZE. LDOX_PETHY: leucoanthocyanidin dioxygenase from P. hybrida; X70786. FLS_PETHY: flavonol synthase from P. hybrida; PIR3:S33510. F3H_HORVU:flavanone-3 hydroxylase from H. vulgare;
SW:FL3H_HORVU. ACCO_PETHY: ethylene-forming enzyme from P. hybrida; L21976. E8_LYCES: 1-aminocyclopropane-1-carboxylic
acid oxidase homolog from L. esculentum; P10967. 2A6: 2A6 mRNA from A. thaliana; X83096. HY6H_HYONI: hyoscyamine 6-hydrolase
from H. niger; SW:HY6H_HYONI. G20H_CUCMA: gibberellin 20-oxidase from C. maxima; GB_CMGIB. G3H_CUCMA: gibberellin
3-oxidase from C. maxima; U63650. G2, 3-CUCMA:gibberellin 2b,3b-hydroxylase from C. maxima; U63650. G7-OXY_CUCMA:
gibberellin 7-oxidase from C. maxima; U61386. VINDOLINE: desacetoxyvindoline 4-hydroxylase from C. roseus (mitochondria) Y09113;
SRG1_ARATH:ORF from A. thaliana; S44261. IDS2: iron deficiency protein from H. vulgare; D15051. ISP7_SCPOM: ORF from S. pombe;
D14064. PROLINE-HYDR:L-proline 3-hydroxylase type II from Streptomyces sp.; TH1. D78337. P4H: prolyl-4-hydroxylase from human;
P13674. LYSY-HYDR: lysyl hydroxylase from human; Q02809. CYSTEINE: cysteine dioxygenase from R. norvegicus, P21816. FERRIC
BINDING: major ferric iron binding protein from N. meningitidis, X53467.

Asp216 and His270 which correspond exactly to the

ligands identified by the genetic and sequence analysis of the C. acremonium and S. jumonjinensis IPNSs
and, unexpectedly, to the penultimate glutamine
residue Gln330 in the molecule. Two solvent molecules complete the coordination shell. Several lines of
evidence indicate that the glutamine ligand in the FeIIIPNS is displaced on binding of substrates. First, the
various spectroscopic studies are consistent with this
idea (Que & Ho 1996); second, site-directed mutagenesis reveals that Gln330 in the A. nidulans IPNS
and the corresponding Gln329 in the S. jumonjinensis
IPNS are not essential for enzyme activity (Landman
et al. 1997; Sami et al. 1997).
This view is now confirmed by the recent report of
the crystal structure at 1.3 of the A. nidulans FeIIIPNS complex with ACV and the oxygen analog NO

(Roach et al. 1997). Because of the instability in air of

the IPNS crystals containing both iron and ACV, the
enzyme was crystallized under anaerobic conditions.
The structure provides for the first time a clear picture
of the interaction of the ACV at the iron center. It verifies that it is the glutamine ligand that is displaced
on ACV binding. The substrate is rigidly anchored
within the active site through ligation of its thiolate
to the ferrous iron, as well as through an extensive
array of interactions of its two carboxylate groups,
and adopts a conformation that approaches that needed
for -lactam formation. In contrast to spectroscopic
studies of the IPNS-FeII-ACV-NO complex, the 3D
structure shows that the NO molecule binds to the iron
center trans to the aspartate ligand (see Figure 2).
As this chapter was about to go to press, the crystal structure of the bacterial S. jumonjinensis IPNS

37

Figure 4. A mechanism for the IPNS catalytic cycle in the conversion of ACV to isopenicillin N, illustrating a possible role for the endogenous
ligands in the active site of the enzyme to function as potential hydrogen acceptors (B); adapted from Blackburn et al. 1995; Roach et al. 1995.

at 2.9 is nearing completion, as well as that of

the FeII-IPNS-ACG (glycine) complex. ACG cannot
serve as a substrate for IPNS to produce penicillins and
severely diminishes the ability of IPNS to cyclize ACV
when pre-incubated with it (A. Scott, pers. comm.). It
was chosen for study since it has been regarded as a
mechanism oriented inhibitor, and because it lacks the
valinyl isopropyl group it is expected to form only the
-lactam ring, thereby leading to an enzyme bound
mono -lactam.
Mechanism of action of IPNS
An understanding of the chemistry by which penicillins are synthesized from tripeptide precursors has
long been sought after (Baldwin & Abraham 1988;
Baldwin & Bradley 1990; Blackburn et al. 1995; Que
& Ho 1996). As outlined above, recent advances in determining the nature of the IPNS structural and functional domains involved in substrate binding and catalytic activity have resulted in considerable progress

towards this goal. Nevertheless, the mechanism is still

obscure primarily because reaction intermediates have
not been isolated.
A scheme for the mechanism of IPNS that takes
into account many of these studies is shown in Figure
4. There is now convincing evidence that the initial
step in the pathway is the ligation of the ACV substrate
to the iron center, which then activates binding of O2
to the vacant site made available by displacement of
the glutamine ligand. This is presumed to be followed
by oxidation of the cysteine thiol and nucleophilic attack of the valinyl nitrogen peptide on the cysteine
-carbon to form the -lactam ring. This process is accompanied by reduction of the iron to an FeII-peroxy
species. Subsequent steps are thought to involve the
formation of the highly reactive iron-oxo Fe(IV)=O
species, through heterolysis of the iron-peroxy intermediate, which can then abstract the -hydrogen atom
from the valinyl isopropyl group followed by closure
of the thiazolidine ring.

38
It has generally been assumed that the role of the
active site ligands in the overall reaction is mainly
to bind the iron. Thus, Roach et al. (1997) point out
that inspection of the active site domain in the crystal
structure of the IPNS-ACV complex fails to reveal any
residues capable of providing general acid-base catalysis, and that the O2 functions as an electron acceptor
in -lactam ring formation, as the source of the reactive iron-oxo species and in removing specific protons
from the substrate.
Nevertheless, genetic studies in which the composition and spatial arrangement of the S. jumonjinensis IPNS histidine and aspartate ligands were altered
(Kreisberg-Zakarin et al. 1998) suggest that the active site ligands may have a functional as well as a
structural role in catalysis, as illustrated in the reaction
scheme shown in Figure 4. Previously we showed that
replacement of each of the ligands by an alanine resulted in the complete loss of activity even though the
mutant enzymes are still able to bind iron (Borovok
at al. 1996, and unpublished results). One possible
explanation is that replacement of either histidine or
the aspartate with alanine causes a change in the electronic structure of the FeII-IPNS-ACV complex which
could affect the ability of the ferrous iron to participate in any one of several steps in the catalytic cycle
(Figure 4). For example, it could affect the change
in the FeII/FeIII redox potential presumably needed
for priming O2 binding. A similar explanation could
account for those mutants in which the aspartate was
replaced by cysteine, histidine or glutamate, among
which only the latter retained a low level of activity; also IPNS mutants in which the aspartate was
exchanged with either of the two histidines were also
totally inactive. In this case the number and type of ligands in the active site were preserved but their spatial
arrangement changed (unpublished results). Possibly,
O2 binding or reactivity depends on the aspartate being
trans to the O2 binding site. Clearly, further studies
will be needed to determine the role of the active site
ligands in catalysis, if at all, and in particular experiments aimed at analyzing the specific structural and
functional defects in active site mutants of the type
described here.

Acknowledgements
We would like to thank Jim Remington, the University of Oregon, and Felix Frolow, Tel-Aviv University,
for communicating preliminary results on the crystal

structure of the Streptomyces jumonjinensis of IPNS,

and the Israel Science Foundation for a grant, No.
488/97.

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