Biochemical Society Transactions (1999) 27
30 Mechanistic insights into substrate turnover and
inhibition of mtallo-p-lactamases
M. J. B. Brown', M. Gilpin'. D.Witty', J. Bateson',
A. J. Pope#
Jlepts. of Molecular Recognition" and Medicinal Chemistry',
SmithKline Beecham, New Frontiers Science Park, Harlow, Essex,
W.K.CM19 5AW
The development of resistance to p-lactam antibiotics i s a constant
threat. The most prevalent mechanism i s the production of plactamases. falling into two main classes with either an active site
serine or a divalent transition metal ion (MpLs). Resistance due to
serine p-lactamases has been addressed by mechanism based serine
traps e.g. clavulanic acid (Augmentinm). Resistance due to MpLs i n
clinical isolates is more recent but the discovery of horizontal transfer
o f the IMP-1 MPL to major pathogens by a mobile gene ( b l n ~ p )
located on an integron-like element represents a wonying trend. Our
interest has been to identify potent inhibitors of MPLs that may be
useful in combination therapy.
produced by B.
The transient kinetics of p-lactam hydrolysis by
jiagilis, have been studied using stopped-flow spectrofluorimetry. For
example, the interaction of Cefoxitin with CfA brought about a large
(278), transient quenching of the intrinsic fluorescence indicating a
major conformational change. The minimal mechanism involves two
binding steps, a weak recognition event then a subsequent tightening.
T h i s has been rationalised in terms of the closure of a flexible loop
over the active site. Catalytic turnover can then occur. We have also
studied ti series of MPL inhibitors based upon Thiorphan, an inhibitor
of the metalloendopeptidase. enkephalinase. Thiorphan shows time
dependent inhibition with weak initial binding reminiscent of
substrate binding. Steady state kinetics indicate that Thiorphan
exhibits a mixed inhibition with an affinity for the E.S binary
complex ten-fold lower than for free E. These mechanistic features
persist through a series of inhibitors based on Thiorphan despite
m j o r improvementsin potency. A tool compound containing a nitroaryl chromophore was prepared which showed marked tryptophan
quenching by fluorescence resonant energy transfer (FRET). Stoppedflow analysis o f this compound allowed dissection of individual rate
constants and subsequent use of microscopic SAR allowed
advancement of the series.
mi,
32
The s t r u c t u r e , f u n c t i o n , and convergent e v o l u t i o n
o f intron-encoded homing endonucleases,
B a r r y L . Stoddard, Melissa J u r i c a , Pat Heath, and
Karen F l i c k
Fred Hutchinson Cancer Research Center. D i v i s i o n o f
Basic Sciences, 1100 F a i r v i e w AVE. N. S e a t t l e WFi
98109 USA
The homing endonuclePses a r e a d i v e r s e f a m i l y o f
p r o t e i n s encoded by open reading frames i n g e n e t i c a l l y
mobile, s e l f - s p l i c i n g i n t r o n s . S i m i l a r endonucleases
have a l s o been i d e n t i f i e d as o p t i o n a l , independently
f c l d e d domains i n s e l f - s p l i c i n g p r o t e i n i n t r o n s ,
termed ' i n t e i n s ' .
These comparatively small enzypec
share t h e a b i l i t y t o recognize and cleave l o n g DNA
s i t e s o f 20 t o 40 bp, and promote t h e l a t e r a l t r a n s f e r
of t h e i r h o s t i n t r o n o r i n t e i n t o these s i t e s b,; a
targetted transposition.
These p r o t e i n s a l s o d'splay
f l e x i b i l i t y o f s i t e - r e c o g n i t i o n , and a r e capeble o f
t o l e r a t i n g changes a t any p o s i t i o n i n t h e t a r g e t DNq
site.
Our l a b o r a t o r y has determined t h e s t r u c t u r e of
r e p r e s e n t a t i v e members o f t w o f a m i l i e s o f homing
endonucleases, both unbound and cOmplexed t c t h e i r
DNA t a r g e t s : I - C r e I ( a LAGLIDADG eneonLClease) ant!
I-PpoJ (a his-cys box endonuclease). The s t r L c t l i r e s
b o t h demonstrate an impressive a b i l l t y of these
p r o t e i n s t o adopt an economical, elongated f o l d dnd
t o form a DNA complex w i t h s u b - s a t u r a t i n g atomic
c o n t a c t s across t h e f u l l l e n g t h of tPe homing s i t e .
The c o - c r y s t a l s t r u c t u r e s i n d i c a t e t h a t t h e er.zyme:
p r o b a b l y fol law tw y e r j d i f f e r e n t s t r u c t l i r a l mechanisms f o r phosphodiester h y d r o l y s i s .
f3CarbonyI Substituted Glutathione Conjugates as Inhibitors
of 0. vdvulus GSTZ.
Peter M. Brophy', Alison M. Cambell', Anna-Maria van Eldick2,
Paul H. Teesdale-SDittleZand Meng F. War@'.
1. Institute of Biological Sciences, University of Wales, Aberystwyth,
Ceredigon, SY23 3DA.
2. Department of Chemistry and Physics, De Montfoil University, The
Gateway. Leicester. LEI QBH.
Parasitic nematodes are responsible for many of the major debilitating
chronic diseases in man, causing more than 1000 million infections world
wide. In particular, the filarial parasite Onchocercu volvulus causes
onchocerciasis; a major cause of preventable blindness and severe
dermatitis in Afnca. Parasites are effectively prdected from the host
immune system by d&ce
enzymes, includmg the glutathione Stransferases (GSTs) which are their major phase I1 detoxification system
and account for up to 4% of the tatal soluble protein. These detoxify
electrophilic compounds, including many anthehtics and cytotoxins
arising from the effector mechanism of the immune response. Thus
inhibition o f parasitic GSTs would severely deplete the parasite defence
system. Two 0.volvulus GSTs have been reported to date, ChGSTl and
ChGST2. We have previously reported the isolation of purified active
recombinant M S T 2 [E. Liebau, et ul (19%) Mol. Biochem. Purusitol.
271. We present here the synthesis of a series of p-carbonyl
substituted glutathione conjugates and data on their inhibition of OvGST2.
Is@values for these conjugates range from 18-5OpM. We also present an
homology model of ChGST2. in common with the A. Suurn GST [E.
Liebau, er ul(1997) Biochem. 1,324. 6591 OvGST2 is shown to possess
an open hydrophobic binding cleft. The inhibition af ChGST2 by these pcarbonyl conjugates is rationalised through molecular modelling. In
particular, an active site tyrosine residue i s almost ideally placed in the
parasite enzymes as a target for binding of a number of GST inhibitors. It
is our hypothesis that suitable derivatisation of p-keto substituted
glutathione Sconjugates will engender tar@ specificity over host
enzymes-
114
31
A39
Structure and Function Studies of Hydroxymethylbilane
Synthase using SRS and ESRF
P.F.Faulder', Y.P.Nieh', J.Raftery', J.Habashi A.Haedene?',
S.McSweeney', F.Schotte4,T.Ursby4, M.Wulff , A.W.Thompson5 and
J.R.Helliwell'
'Dept. of Chemistry, University of Manchester; 'Dept. of Chemistry,
Universityof Basle; 3SRS. Daresbury; 4ESRF, Grenoble 'EMBL, Grenoble
Hydroxymethylbilane synthase (EC 4.3.1.8, HMBS). also known as
porphobilinogen deaminase, is the third enzyme in the biosynthetic
pathway of the tetrapyrroles which include haem, chlorophyll, vitamin 812
and similar pigments. HMBS catalyses the polymerisation of four
molecules of porphobilinogen to form hydroxymethylbilane. A
dipyrromethane cofactor is present in the HMBS holoenzyme and is
covalently attached to a cysteine side-chain (Cys242). There are four
covalent enzyme-substrate intermediate complexes existing in the
catalytic pathway, i.e. ES1, ES2, ES3, ES4. Previously, a reduced, active
selenomethionine form of the holoenzyme has been solved to 2.4A using
the MAD method (Acta Cryst D. submitted) principally at SRS station 9.5
and subsequently also at ESRF BM14. Time-resolved Laue diffraction
has since been employed at ESRF to study the enzyme structure (K59Q
mutant) during catalysis (Faraday Trans, in the press). Experimental
difference maps revealed an elongated peak near the active site, most
prominent at 2 hours, commencing at the position of ring 2 of the oxidised
cofactor (the putative binding site for substrate) and directly above the
critical Asp84 carboxyl side chain implicated in the first ring coupling step.
The density then extends past residues that are known from protein
engineering to affect later stages of the catalysis, and out into open
solvent. In order to improve the 'static' protein structure, and to establish
freeze trapping conditions, since the time-evolutionof this irreversible (i.e.
in terms of the colourless to red colour change) reaction in the crystal has
now been established from the Laue diffraction,cry0 temperature data
has been collected on the reduced, active wild-type HMBS holoenzyme to
1.65A at ESRF (ID09). The refinement of the cryo-model, based on the
wild-type HMBS Laue structure as the initial starting model, is in progress
and currently has R-factor=24.0% and Rfree=29.2%. Freeze trapping of
key stages in this irreversible process in the crystal will allow highly
complete diffraction data (Laue or monochromatic) to be collected at
'leisure'. Explorationof putative domain-domain movements of the protein
will be explored by time-resolved synchrotron solution X-ray scattering.