4th EucheMs chemistry congress

Monday, 27-Aug 2012
s704
chem. Listy 106, s587–s1425 (2012)
life sciences
Biocatalysis session – i
o - 0 5 2
SoLution StruCtureS And ModeLS deSCriBinG
the thioredoxin SySteM froM
MyCoBACteriuM tuBerCuLoSiS
t. neuMAnn 1 , A. oLSon 2 , S. CAi 3 , d. SeM 4
1 Marquette University, Chemistry, Milwaukee Wi, USA
2 North Carolina State University, Department of Structural and
Molecular Biology, Raleigh NC, USA
3 Marquette University, Chemistry, Milwaukee WI, USA
4 Concordia University Wisconsin, School of Pharmacy, Mequon
WI, USA
Mycobacterium tuberculosis (M. tb) resists oxidative killing
in part by using the thioredoxin (Trx) system. [1] Trx catalyzes
thiol-disulfide exchange reactions using redox active cysteine
thiols to reduce disulfides of other essential proteins, including
metabolically essential enzymes. [2–4] Oxidized Trx is then reduced
by thioredoxin reductase (TrxR) in an NADPH dependant
reaction. [5] The M. tb Trx system consists of three Trx’s (TrxA,
TrxB, and TrxC) and one Trx reductase (TrxR). TrxR is essential
for survival. TrxB and TrxC are known substrates of TrxR. [1] TrxA,
meanwhile, has been reported to not bind to TrxR and to possibly
be “cryptic”. [1] The M. tb Trx system is dissimilar to the human Trx
system (25–35% indentity) such that inhibitor specificity for the
M. tb Trx system should be obtainable. Thus, the M. tb Trx system
appears to be a viable drug target. [6]
The objective of this study was to structurally characterize
oxidized and reduced Trx’s. Solution structures have been
calculated using standard NMR solution experiments. [7] Our
studies indicate that TrxA is well-folded in both oxidized and
reduced states. Structures of the individual Trx’s and binding
models of the TrxN(N = A, B, or C)-TrxR, constructed from NMR
titrations of each 15N enriched TrxN and unlabeled TrxR, are
discussed. [7] These binding models show an empty pocket between
the Trx and the TrxR, that is targeted for structure-based design
of uncompetitive inhibitors.
references:
1. Akif, M., et. al. J. Bacteriol. 2008, 190, 7087-7095.
2. Gleason, F. K., et. al. FEMS Microbiol. Lett. 1988, 54,
271-297.
3. Ortenberg, R., et. al. Proc. Natl. Acad. Sci. U. S. A. 2004,
101, 7439-7444.
4. Powis, G., et. al. Pharmacol. Ther. 1995, 68, 149-173.
5. Williams, C. H., et. al. Eur. J. Biochem. 2000, 267,
6110–6117.
6. Zhang, Z., et. al. Arch. Biochem. Biophys. 1999, 363,
19–26.
7. Olson, A.L., et. al. Biochemistry. (under review)
Keywords: Mycobacterium tuberculosis; nuclear magnetic
resonance; structural biology; thioredoxin system;
Biocatalysis session – i
4 th EucheMs chemistry congress
o - 0 5 3
the LiGht MAKeS it worKS. MoLeCuLAr
reACtion dynAMiCS inveStiGAtion At
uLtrAfASt tiMe SCALe on
nAdPh:ProtoChLoroPhyLLide
oxidoreduCtASe
A. GArrone 1 , S. fey 1 , J. SChäfer 2 , G. herMAnn 1 ,
B. dietzeK 2
1 Friedrich-Schiller Universität Jena, Institut für Biochemie und
Biophysik, Jena, Germany
2 Institut für Photonische Technologien - IPHT, Ultrafast
Spectroscopy Group, Jena, Germany
The light driven NADPH:protochlorophyllide
oxidoreductase (POR) is a key enzyme of chlorophyll
biosynthesis in angiosperm and cyanobacteria. POR’s unique
requirement of light to became active, makes the enzyme an
attractive model to study the dynamics of enzymatic reaction in
real time. It catalyzes one of the later steps in the chlorophyll
synthesis pathway: the light-dependent reduction of
protochlorophyllide (PChlide) to chlorophyllide by trans addition
of hydrogen across the C17-C18 double bond in the porphyrin
ring.
The ternary complex NADPH-POR-PChlide has been
reconstituted in vitro and the initial reaction steps were followed
using various spectroscopic tools such as femtosecond/picosecond
time-resolved absorption and fluorescence spectroscopy in the
UV/vis spectral range. To highlight the mechanistic interplay
between the protein moiety and enzyme function, point mutations
were introduced into the catalytic site.
Two point mutants, have been produced: an aspartate was
substituted by methionine and a serine by alanine. Here we report
our first results on three POR isoforms: POR A and POR B from
barley (Hordeum vulgare) and POR from Synechocystis. We
observed that the fluorescence decay time of PChlide when bound
to the protein WT (wildtype) is about 1 ns longer than in solution.
We compared the kinetic parameters of the photoreaction with the
fluorescence decay time for the different isoforms and mutants.
We also discuss the role of these substitutions in the substrate
binding site and their effect on the H-bond network, which we
suggest to play a prominent role in first fast reaction steps.
Keywords: Enzyme models; Mutagenesis; Time-resolved
spectroscopy; Oxidoreductases;
AUGUst 26–30, 2012, PrAGUE, cZEcH rEPUbLIc