Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-ACP synthase III mtFabH

24 Pages·2005·1.44 MB·English

JBC Papers in Press. Published on July 22, 2005 as Manuscript M413216200
Probing the mechanism of the Mycobacterium tuberculosis β-ketoacyl-ACP
synthase III mtFabH: factors influencing catalysis and substrate specificity*

Alistair K. Brown1,2,§, Sudharsan Sridharan3,§, Laurent Kremer4 Sandra Lindenberg2, Lynn G. Dover2,
,
James C. Sacchettini3 and Gurdyal S. Besra2

From the 1School of Cellular and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon
Tyne, NE2 4HH, UK, 2School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT,
UK, 3Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128,
USA, 3Laboratoire de Dynamique Moléculaire des Interactions Membranaires, CNRS-UMR 5539, Université
de Montpellier II, Case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France.

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis causes more human deaths
Mycobacterium tuberculosis cell wall. These α-alkyl, than any other single infectious organism with an
β-hydroxy fatty acids are formed by the estimated 8 million new tuberculosis (TB) cases and 2
condensation of two fatty acids, a long meromycolic million fatalities per annum (1). An apparent synergy
acid and a shorter C -C fatty acid. The has evolved between the epidemiology of AIDS and
24 26
component fatty acids are produced via a TB, with the continuing HIV pandemic intensifying the
combination of type I and II fatty acid synthases growing problems in tuberculosis control (2-4). Current
(FAS) with FAS-I products being elongated by FAS- anti-tubercular drug regimens are lengthy, usually
II towards meromycolic acids. The β-ketoacyl-ACP lasting up to 6 months and include three or four D
o
synthase III (KAS-III) encoded by mtfabH (mtFabH) components. Consequently, poor patient compliance is wn
links FAS-I and FAS-II, catalyzing the condensation one of the major contributing factors in the loa
d
of FAS-I-derived acyl-CoAs with malonyl-Acyl development of multidrug resistant-TB (MDR-TB) (5). ed
carrier protein (ACP). The acyl-CoA chain length Given the current backdrop of emerging MDR-TB (6), fro
m
specificity of mtFabH was assessed in vitro; the tuberculosis treatment is entering a new and h
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natural partner, than with E. coli ACP. The w
The low permeability of the unusual lipid-rich cell wall .jb
enzyme’s ability to use ecACP suggests that a c
of M. tuberculosis is a major contributor to its intrinsic .o
ysiemt iilta ris mcloeadre tohfa tb iunndiiqnuge ifsa clitkoerlsy inwhitehre nbot ttho AACcpPMs, rceosmisptalenxc es ttrou cmtuorset, cwomhimcho ncloyn-tuasiends asenvtiebriaolt iccosm (7p)o. nTehnitss by grg/
modulate the substrate specificity of mtFabH. u
essential to both mycobacterial viability and e
s
Mutation of proposed key mtFabH residues was pathogenicity, also represents arguably the most t o
n
used to define their catalytic roles. Substitution of successful target of antimycobacterial chemotherapy Ja
supposed acyl-CoA binding residues reduced (8). The most striking feature of the mycobacterial cell nua
transacylation, with double substitutions totally ry
wall are the α-alkyl, β-hydroxy fatty acids termed 8
abrogating activity. Mutation of Arg46 revealed its mycolic acids, which are esterified to the non-reducing , 20
more critical role in malonyl-AcpM decarboxylation termini of the arabinogalactan-peptidoglycan cell wall 19
than in acyl-CoA binding role. Interestingly, this
core and form the inner leaflet of the cell wall
effect was suppressed intragenically by
permeability barrier (8,9). Although the longer
Arg161(cid:198)Ala substitution. Our structural studies
meromycolate chain (up to 56 carbon atoms) of the
suggested that His258, previously implicated in
mycolic acids are modified (10), its length is essentially
malonyl-ACP decarboxylation, also acts as an
provided via conventional fatty acid biosynthesis. Upon
anchor-point for a network of water molecules that
completion it is condensed with a C -C fatty acid,
we propose promotes deprotonation and 24 26
which provides the α-alkyl branch of the mature
transacylation of Cys122.
mycolic acid (9,11,12). Mycobacteria are unusual in

§ These authors contributed equally. that they possess both known types of fatty acid
Author for correspondence: Gurdyal S. Besra. Tel: +44 121 synthase (FAS) (10); a mammalian-type FAS-I, which
415 8125. Fax: [phone]. Email: carries all of the necessary enzymatic activities and
[email protected]

* This work was supported in by The Medical Research
The abbreviations used are: ACP, acyl carrier protein; BSA,
Council, The Wellcome Trust and the National Institute of
bovine serum albumin; CoA, Coenzyme A; DTT,
Health (NIH, USA). GSB is a Lister-Jenner Research Fellow.
dithiothreitol; ecACP, E. coli ACP; FAS, fatty acid synthase;
The costs of publication of this article were defrayed in part
IPTG, isopropyl-1-thio-β−D-galactopyranoside; KAS, β-
by the payment of page charges. This article must therefore be
ketoacyl-[acyl carrier protein] synthase; MCAT, malonyl-
hereby marked "advertisement" in accordance with 18 U.S.C.
CoA:ACP transacylase; mtFabD, Mycobacterium tuberculosis
Section 1734 solely to indicate this fact.
MCAT; mtFabH, Mycobacterium tuberculosis KAS-III; PCR,
polymerase chain reaction; PEG, polyethylene glycol.
1
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
carrier functions on a single polypeptide (13), and a obtained from Amersham Biosciences, [114C]C -CoA
10
bacterial type FAS-II, in which separate enzymes (specific activity, 55 Ci/mol) from American
interact with a discrete acyl carrier protein (ACP) that Radiolabeled Chemicals Inc. Restriction enzymes and
carries the growing fatty acyl chain between their active T4 DNA ligase were obtained from Roche Molecular
sites (14). In mycobacteria and related genera, FAS-I Biochemicals. Vent DNA polymerase was from New
conducts the de novo synthesis of intermediate length England BioLabs and pET28a from Novagen.
(principally C and C ) fatty acids. Mycobacterial EcoScintA was purchased from National Diagnostics.
16 24
FAS-II, however, is incapable of de novo fatty acid Centricon YM-10 filtration units were obtained from
synthesis (15-18). Its ability to elongate C - and C - Millipore. E. coli C41(DE3) (27), obtained from Avidis
14 16
CoA primers has been demonstrated (11,19-21) and (France), was used for protein production in this study.
current hypotheses suggest that FAS-I products are M. tuberculosis H37Rv DNA was obtained from Dr J.
elongated via FAS-II to form meromycolates (9). T. Belisle and the NIH Tuberculosis Research Materials
and Vaccine Testing Contract at Colorado State
In both FAS-I and FAS-II, fatty acyl elongation is
University. All other chemicals were reagent grade or
initiated by a condensation reaction in which malonyl-
better and obtained from Sigma-Aldrich.
ACP is decarboxylated and the resulting carbanion
attacks an enzyme-linked acyl thioester derived from an Cloning of mtfabH─Amplification of the mtfabH gene
acyl-CoA primer (11,13,20). In FAS-II the initial was performed with Vent DNA polymerase (New
condensation reaction is carried out by β-ketoacyl-ACP England Biolabs) using M. tuberculosis H37Rv
synthase (KAS)-III (22), however subsequent FAS-II chromosomal DNA and the following primers: fabH-
cycles are initiated by acyl-ACP primed β-ketoacyl- ATG 5‘-gta tga cgg aga tcg cca cga cc-3’ and fabH-
ACP synthases, KAS-I and KAS-II (KasA and KasB Cterm 5’-gga att cgc gcg ctc aac cct tc-3’. The PCR D
respectively in M. tuberculosis) (11,17,22,23). product was then directly ligated into pET28a ow
n
(Novagen), which had been cut by NdeI and filled-in lo
We recently identified a KAS-III (mtFabH) enzyme in with Klenow enzyme (Roche Biochemicals), thus ade
Mrec. otmubbeirncaunlto siesn. zUymnlei kee xEt.e ncdoeldi KiAntSe-rImIIe, dtihaete pucrhifaiiend- gsuivbisnegq ureisnet lyto upsEeTd2 8tao- mttrfaanbsHfo. rmTh isE .p lacsomli idC w41as(D thEe3n) d from
length acyl-CoA primers rather than acetyl-CoA and h
enabling the production of the recombinant mtFabH ttp
was proposed to represent a pivotal link between proteins. ://w
mycobacterial FAS-I and FAS-II (20). The elucidation w
of the crystal structure of this KAS-III (mtFabH) Site-directed mutagenesis of mtFabH─A series of site- w.jb
revealed a similar topology and active-site architecture directed mutants were obtained using pET28a-mtfabH c.o
tsoe vtehraatl ffarcotmor sE wscehree rpircohpiao secdo ltio (iencfFluaebnHc)e t(h2e4 -s2u6b)s traantde aMs utatgeemnpelsaitse Kfoitr (tShter ataQgeunikeC) haancgceo rdiSnigte -Dtoir ecttehde byrg/
g
specificity of mtFabH. In both ecFabH and mtFabH, manufacturer's instructions with the following primers: ue
s
similar acyl-CoA/malonyl-ACP-binding channels C122A: sense 5’-gat ctt tcg gcg ggg gcc gcc gga ttc gga t o
n
[channel 1, (26)] lead from the surface to the buried tat gcg-3’ and antisense 5’-cgc ata tcc gaa tcc ggc ggc J
a
n
active site catalytic triad. The binding of acyl-CoA ccc cgc cga aag atc-3’ ; H258A: sense 5’-gac gtg ttc gtc u
a
substrates was proposed to be facilitated by interactions cct gct cag gcc aat agc cgc atc-3’ and antisense 5’-gat ry 8
of the CoA moiety with Trp42, Arg161 and Arg46 (26). gcg gct att ggc ctg agc agg gac gaa cac gtc-3’ ; N289A: , 2
0
The mycobacterial enzyme contains a second sense 5’-atc gag cac acc gga gcc acc tcg gcg gcc tcc att- 19
hydrophobic channel [channel 2, (26)] leading from the 3’ and antisense 5’-aat gga ggc cgc cga ggt ggc tcc ggt
active site. In ecFabH, this second channel is blocked gtg ctc gat-3’ ; W42A: sense 5’-gac tcg tcc gac gag gcg
by a Phe residue (a permissive Thr in mtFabH), atc tac acc cga acc gcc-3’ and antisense 5’- ggc ggt tcg
effectively limiting its substrate specificity to acetyl- ggt gta gat cgc ctc gtc gga cga gtc-3’ ; R46A: sense 5’-
CoA by preventing the binding of longer acyl-CoA gag tgg atc tac acc gca acc ggc atc aag acc cgc-3’ and
primers. In mtFabH, channel 2 is capped by 4 amino antisense 5’-gcg ggt ctt gat gcc ggt tgc ggt gta gat cca
acid residues (Ser213-Gly-Arg-Pro216), which was ctc-3’ ; R161A: sense 5’-ata gac atg tac gac gcc ggc aac
proposed to limit its acyl-CoA primer chain length to tgc ttc atc ttc-3’ and antisense 5’-gaa gat gaa gca gtt gcc
16 carbons (26). ggc gtc gta cat gtc tat-3’ ; T97F: sense 5’-acc cat ttc ctg
caa ttc ccg ccg gcc gcc cca atg-3’ and antisense 5’-cat
In this study we have used site–directed mutagenesis to
tgg ggc ggc cgg cgg gaa ttg cag gaa atg ggt-3’.
analyse the contribution of specific residues to the
KAS-III activity of mtFabH. Each mutant has been The doubly-substituted mtfabH alleles were constructed
characterized through analyses of the component by digesting the pET28a-mtfabH R161A plasmid with
transacylation and malonyl-ACP decarboxylation part- StuI and EcoRI which cleaves the plasmid DNA such
reactions to gain further insight into the role played by that the 5’ end of the mtfabH gene is removed. The
their substituted residues. second mutations were then incorporated by replacing
the excised DNA with analogous fragments purified
EXPERIMENTAL PROCEDURES
from similar digests of pET28a-mtfabH Arg42(cid:198)Ala
Materials─[2-14C]malonyl-CoA (specific activity, 55.0 and pET28a-mtfabH Arg46(cid:198)Ala. The identity of all
Ci/mol) and Amplify fluorographic reagent were mutant alleles was confirmed by nucleotide sequencing.
2
Purification of the His -tagged holo-AcpM, mtFabH began to reduce after 2 min incubation we chose a 1
6
and mutants─Recombinant proteins were generated and min time point to compare mtFabH and its mutants.
purified as described previously (20,28). Purified His -
6 Malonyl-ACP decarboxylation Assay─The method
tagged mtFabH, and mutants thereof, were dialyzed
used by McGuire et al. (30) was adapted to measure the
extensively against 20 mM Tris.HCl, pH 7.6, 1 mM β-
decarboxylation of malonyl-ACP by mtFabH. In this
mercaptoethanol and were concentrated using
assay malonyl-ACP was synthesized from malonyl-
Centricon YM-10 filter units and stored in 50 %
CoA and holo-ACPs in situ using mtFabD. Synthesis of
glycerol at -80°C.
malonyl-ACP was accomplished using 25 mM
mtFabH assay─ The assay developed for ecFabH (29) potassium phosphate buffer (pH 8), 12 µM ACP, 8.2
was previously modified to measure the activity of µM malonyl-CoA, 45 nCi [2-14C]malonyl-CoA
mtFabH with radio-labeled [2-14C]malonyl-CoA (20). (specific activity 55 Ci/mol), 1 mM DTT, and 0.3 µg of
As previously, the assays contained 50 µM holo- mtFabD preparation per assay. mtFabD and the
ACP/AcpM, 1 mM β-mercaptoethanol, 0.1 M sodium appropriate ACP were pre-incubated with DTT for 60
phosphate buffer, pH 7.0, 50 µM malonyl-CoA, 45 nCi min on ice. After equilibrating for 5 min at 25°C, the
[2-14C]malonyl-CoA (specific activity 55 Ci/mole), reaction mixture was transferred to ice. For a 100 µl
12.5 µM acyl-CoA primer and mtFabD (0.3 µg of decarboxylation reaction, 83 µl of reaction mixture was
protein) in a volume of 50 µl. The mtFabD protein was dispensed to a microcentrifuge tube with 17 µl (4.25
added to generate the malonyl-ACP substrate for the µg) of mtFabH or mutant. After a further 10 min at
reaction in situ. A mixture of ACP, 1 mM β- 25°C the reaction was quenched by the addition of 900
mercaptoethanol, and the buffer was incubated at 37°C µl of ice-cold 10 % (w/v) trichloroacetic acid (4°C) and
for 30 min to ensure complete reduction of ACP, and D
50 µl of 2 mg/ml BSA and incubated on ice for 30 min. o
then the remaining components (except mtFabH) were The solution was centrifuged at 27,000 x g for 30 min, wn
added. The mixture was then dispensed into and the protein pellets were resuspended in 200 µl 50 load
microcentrifuge tubes and the reaction initiated by the ed
addition of 0.5 µg mtFabH. The reaction mixture was mM Tris.HCl (pH 6.8). Samples (20 µl) were taken and fro
analysed by electrophoresis on 13.3 % PAGE gels m
held at 37°C for 40 min. containing 1 M urea (30). The gels were fixed, soaked http
After this point our assays differed from those in Amplify fluorographic reagent (Amersham ://w
previously described (20,29). Here we quenched the Biosciences), dried and visualised by autoradiography ww
reactions by adding 5 mg/ml NaBH in 100 mM using Kodak X-Omat film exposed for 72 hours at - .jb
4 c
K HPO , 100 mM KCl, 30 % tetrahydrofuran resulting 80°C. .o
2 4 rg
in the liberation of β-ketoacyl groups from their Crystallization and structure determination of by/
respective thioesters as acyl-1,3-diols. These products Arg46Arg161(cid:198)Ala and Trp42Arg161(cid:198)Ala mutants. gu
e
were extracted three times with 1 ml water-saturated The Arg46Arg161(cid:198)Ala and Trp42Arg161(cid:198)Ala st o
toluene, the extracts were pooled and washed with an mutant proteins were purified using nickel affinity and n J
equal volume of water. The organic component was gel filtration chromatography for crystallization anu
transferred to scintillation vials and the solvent purposes. Both the mutants were crystallized using ary
removed by evaporation, 5 ml EcoScintA was added microbatch-under-oil method at room temperature. The 8, 2
and the radio-labelled products quantified by liquid Arg46Arg161(cid:198)Ala mutant was crystallized by mixing 01
9
scintillation counting. 3 µl of about 20 mg/ml protein in 20 mM Tris.HCl pH
Transacylation Assay─We monitored the transacylation 7.5, 0.5 M NaCl, 2 mM β-mercaptoethanol, 10 %
of mtFabH and mutants using a modification of the glycerol with 1 µl of Hampton Research Crystal Screen
assay developed for KAS-I (30) in which we utilized I, condition 41 (0.1 M HEPES pH 7.5, 10 % v/v
the His tag of our recombinant enzymes to facilitate isopropanol, 20 % w/v PEG 4000). The
6
removal of unbound radiolabel. Acyl transfer was Trp42Arg161(cid:198)Ala mutant was crystallized by mixing
assessed using [14C]C -CoA to follow transfer of the 2 µl of 20 mg/ml protein in 20 mM Tris.HCl pH 7.5,
10
acyl chain from acyl-CoA to Cys122 of mtFabH. The 0.5 M NaCl, 2 mM β-mercaptoethanol, 10 % glycerol
reaction mixture consisted of 50 mM Tris.HCl pH 7.5 with 1 µl of Hampton Research Crystal Screen I,
(Buffer A), 250 pmol [14C]C -CoA (14.6 nCi), and 4 condition 30 (0.2 M ammonium sulfate, 30 % w/v PEG
10
µg mtFabH/mutant (110 pmol) in volume of 60 µl. The 8000).
reaction was held at ambient temperature (~20°C) for
X-ray data were collected at the synchrotron beamline
up to 15 min, 200 µl of Ni2+ charged chelating 14 of the Advanced Photon Source, Argonne National
Sepharose fast flow chromatography media (50 % Laboratory, Illinois. The data were processed, scaled
slurry in buffer A) added and incubation continued for a and merged using the HKL2000 software package (31).
further 5 minutes. The slurry was diluted by adding 300 The native mtFabH structure was solved using
µl buffer A and unbound C10-CoA was then removed molecular replacement. A structural model for the
by filtration (cellulose nitrate, 0.45 µm). The beads mtFabH sequence generated by the Swiss Model server
were washed with 20 ml buffer A before the filters (32) was used as the search probe for cross-rotation and
were removed to vials for liquid scintillation counting translation searches in CNS (33). Simulated annealing
with 5 ml EcoScintA. As the rate of label retention was implemented during initial rounds of refinement
3
using CNS. The program O (34) was used for building for acyl primers to those reported for other FabH
models. The model was refined to a final R-factor of enzymes (40,41).
21.2 % and R-free factor of 26.5 % after iterative model
KAS-III activity of mtFabH mutants─The availability of
building and refinement. For the determination
the mtFabH crystal structure allowed us to target
Arg46Arg161(cid:198)Ala and Trp42Arg161(cid:198)Ala mutant
specific residues for site-directed mutagenesis
structures, molecular replacement solutions were
facilitating a biochemical analysis of their individual
obtained with the structure of native mtFabH dimer as
contributions to KAS-III activity. Three groups of
the model using Molrep program (35) in the CCP4 suite
amino acid residues were chosen for analysis, classified
(36). Models were built using Xtalview software (37)
according to their predicted functional roles; residues
and structure refinement with simulated annealing
contributing the catalytic triad (Cys122, His258 and
during the initial rounds was done with CNS suite (33).
Asn289), those implicated in acyl-CoA binding (Trp42,
Non-crystallographic symmetry was applied during all
Arg64 and Arg161) and Thr97, which is thought to
rounds of refinement. Several rounds of model building
influence the acyl-CoA primer chain length specificity
and refinement and addition of waters resulted in final
of the enzyme.
R-factor/R-free factor values of 22.5 %/ 27.9 % for the
Trp42Arg161(cid:198)Ala mutant and 24.8 %/ 27.8 % for the The KAS-III activity of each mutant was determined
Arg46Arg161(cid:198)Ala mutant. Unbiased electron density (Table III). As expected, mutation of the catalytic triad
maps for the mutant structures were also calculated residues completely abrogated activity confirming that
using the TB Bias Removal Server (38). these residues are all vital for the catalysis of the
Crystallographic data and refinement statistics are condensation reaction. The KAS-III activity of the
given in Table I. Thr97(cid:198)Phe mutant was totally abrogated. The single
substitutions Trp42(cid:198)Ala, Arg46(cid:198)Ala and D
RESULTS ow
Arg161(cid:198)Ala reduced mtFabH activity to 21.56, 7.32 n
lo
Purification of mtFabH and mutants─The integrity of and 69.66 % of the wild-type enzyme respectively a
d
e
the mutant mtFabH alleles was confirmed by complete confirming a non-essential role in catalysis consistent d
nucleotide sequencing. Over-expression of these alleles with their predicted function as contributors to the fro
m
in E. coli C41(DE3) yielded large quantities of His - binding of acyl-CoA primers (26,42). The doubly- h
tsaogmgee dp rmeuptaarnatt iopnrso tecionn t(a~in1e0d mmgi/nLo rc uclotunrtea)m. iAnaltnhtos,u gi6nh Asurbgs4ti6tAutregd1 61(cid:198)mAultaa ntssh oweTdr p4f2uArthrge1r 61r(cid:198)edAuclati ons anind ttp://ww
mafofisnti tcya secsh proumrea tporgortaepinh yw aso no btNaiin2+e-dc hbayr gmedet alH-cihse-tlraatpe activity, to 0.24 and 0.31 % of mtFabH respectively. w.jbc
Transacylation of mtFabH and mutants─The .o
chelating Sepharose columns (Figure 1). rg
contribution of a particular residue to the overall KAS- b/
y
Acyl Carrier Protein-dependent acyl-CoA chain length III activity can be more accurately defined by g
u
preference of mtFabH─The genome of M. tuberculosis considering the performance of its substituted mutant in es
H37Rv contains three putative ACP genes. One of the component part-reactions. In the first transacylation t on
J
these, acpM, lies within a cluster of genes encoding reaction, the acyl chain is transferred from a CoA- an
u
several FAS-II components suggesting its synthesis is linked primer to form a thioester with the active site ary
co-regulated with these enzymes. Moreover, antisera Cys122 residue Sγ. We analysed the transacylation 8, 2
raised against AcpM inhibits in vitro modification of activity of mtFabH, sampling the reaction over a 15 min 0
1
9
growing meromycolate precursors thereby providing period (Figure 2). We selected the C -CoA substrate
10
experimental evidence that implicates AcpM as the as it represents a very good fatty acyl primer in our
carrier throughout meromycolate extension and KAS-III assays when using ecACP, i.e. in the absence
modification (39). In order to determine whether its of the substrate specificity modulating effects of AcpM.
ACP partner influences KAS-III activity, we measured After the first 2 minutes, the rate of label retention
[14C]malonate incorporation in a fatty acyl primer began to decrease but the transacylation of the protein
extension assay using a range of acyl-CoA primers, continued throughout the sampling period resulting in
mtFabH and either AcpM or the E. coli ACP (ecACP) 23 % saturation after 15 min. We then analysed the
as carriers. Quantification of [14C]malonate transacylation activities of the various mutants (Table
incorporation revealed an ACP-dependent shift in the III and supplementary data, Figure S1). As expected, no
acyl-CoA primer chain length specificity exhibited by transacylation was evident with the Cys122(cid:198)Ala
the enzyme (Table II). With ecACP, the optimal fatty substituted mutant, consistent with the loss of the active
acyl primer for mtFabH was found to be C -CoA; both site sulfydryl group necessary for acyl thioester
12
C - and C -CoAs were also efficiently extended but formation. All of our other mutants, apart from the
8 10
longer acyl-CoAs represented poor primers (Table II). substitution of the supposed acyl-CoA binding residue
In contrast when AcpM was used as the carrier, Trp42(cid:198)Ala, exhibited decreased transacylation
[14C]malonate incorporation continued to increase with activities. Although the mutants substituted at the
acyl chain length to reach an apparent maximum with active site residues His258(cid:198)Ala and Asn289(cid:198)Ala
primers ranging in length from C -CoA to C -CoA were both capable of transacylation, the extent of
14 20
(Table II). The apparent K for C -CoA was calculated protein acylation was reduced suggesting that neither
M 12
as 2.3 µM (data not shown) revealing a similar affinity residue is essential for, but both make minor
4
contributions to the transfer of the acyl moiety. The of the Arg46 mutation by the Arg161(cid:198)Ala
His258(cid:198)Ala substitution had a more profound effect substitution.
on acyl transfer than did individual substitutions in the
Crystal structures of Arg46Arg161(cid:198)Ala and
proposed acyl-CoA binding residues (Trp42(cid:198)Ala,
Trp42Arg161(cid:198)Ala mutants─Dramatic effects on
Arg46(cid:198)Ala and Arg161(cid:198)Ala) and was comparable
catalysis were observed with both doubly-substituted
with the double arginine-substituted mutant. However,
mutants. To ascertain whether these effects were due
the other doubly-substituted mutant Trp42Arg161(cid:198)Ala
simply to changing the two side chains rather than more
did exhibit a poor transacylation activity, presumably
profound consequential structural rearrangements, we
due to a low affinity for the acyl-CoA substrate.
determined the X-ray crystal structures of wild type
Decarboxylation of malonyl-ACP by mtFabH mutants─ mtFabH, and the Arg46Arg161(cid:198)Ala and
After transacylation, the deacylated CoA leaves the Trp42Arg161(cid:198)Ala mutants to resolutions of 2.7 Å, 2.0
active site allowing the acyl-pantetheine arm of Å and 2.5 Å respectively (see Table 1). The structures
malonyl-AcpM to enter via the same canal [channel 1, of the mutants are virtually identical to that of the
(26)]. The subsequent decarboxylation of the malonyl native mtFabH determined earlier (PDB code 1M1M).
moiety produces a carbanion that displaces the enzyme- The RMS deviations when 325 equivalent C-alpha
linked thioester and thus extends the acyl-ACP. atoms of the mutant structures are superposed over that
Electrophoresis on conformationally-sensitive, urea- of the native is 0.3 Å for the Arg46Arg161(cid:198)Ala
containing PAGE gels has previously been used to mutant and 0.4 Å for the Trp42Arg161(cid:198)Ala mutant.
separate variously substituted ACPs (30) and was
The disposition of the active site residues is almost the
applied here to analyse the ability of the mtFabH
same in all three structures except for the orientation of
mutants to decarboxylate radio-labelled malonyl-ACPs.
the side chain of His258. While the side chain has the D
As the decarboxylation of malonylated ecACP and ow
same orientation in the native mtFabH structure n
AcpM proceeded similarly when using mtFabH and the lo
(1M1M) and Arg46Arg161(cid:198)Ala mutant, in the a
radiolabelled species behaved similarly on de
Trp42Arg161(cid:198)Ala mutant structure and the native d
electrophoresis (Figure 3), AcpM was used in all of our mtFabH structure described by Scarsdale et al. (1HZP, fro
analyses. Without mtFabH, radioactive species m
(26)), the plane of His258 imidazole ring is tilted by h
icvnoistrrirobedlseup coen(ddF,i ingagu rnteoe wm3 a)rl.ao dniyHoll-oaCwboeeAlvlee dar n dspw mehcaeilenos n yimsl- tAFocbapbseMHrv aerdies, acabtoaulyt ti3c 0h°i.s tiTdhinues inth teh ecshe aanngde thine Aorrgie4n6tAatrigo1n6 1o(cid:198)f Athlea ttp://ww
corresponding to acetyl-AcpM, the product of the mutant structures is likely induced by ligand binding in w.jb
the active site channel as described below. c
decarboxylation of malonyl-AcpM. In order to quantify .o
rg
malonyl-AcpM decarboxylation, we used densitometry In the Arg46Arg161(cid:198)Ala mutant structure, fragmented b/
y
of autoradiograms to determine the decarboxylation electron densities that extend in length between 3 and g
u
activity of the individual mutants relative to mtFabH 12 Å in both the monomers are seen in both the acyl- es
(Figure 3). When using Arg161(cid:198)Ala, binding channel and a second putative acyl-binding t on
J
Trp42Arg161(cid:198)Ala, Arg46Arg161(cid:198)Ala and channel proposed by Scarsdale et al. (26) in their an
u
His258(cid:198)Ala mutants a novel radiolabelled product description of the mtFabH crystal structure (1HZP). In ary
migrating between malonyl-AcpM and acetyl-AcpM that article, these channels have been labeled binding 8
, 2
was observed. We have attempted to characterize this channels 1 and 2 respectively and a lauroyl group has 0
1
9
novel species using mass spectroscopy but to date it been modeled in channel 1 and a myristoyl group in
remains unidentified. All of the mutants produced in channel 2. Superposition of the 1HZP structure and
this study exhibited decreased malonyl-AcpM Arg46Arg161(cid:198)Ala mutant structures suggests that in
decarboxylation activities (Figure 3 and Table III). The channel 1 a lauroyl group maybe positioned in a similar
catalytic triad residues His258(cid:198)Ala and Asn289(cid:198)Ala manner in both the structures. In Figure 4 a C acyl
7
had previously been implicated in the decarboxylation group has been modeled in this density. Acyl binding in
of the malonyl-AcpM (26). Consistent with this, the this channel could possibly cause conformational
decarboxylation activity of His258(cid:198)Ala was changes of the active site residues such as that observed
completely abrogated whereas that of the Asn289(cid:198)Ala for His258 above. Part of the density in channel 1 has
mutant was markedly reduced. These observations been modeled as a water molecule as described below.
strongly suggest that His258 is essential for this part- The density seen in the binding channel 2 extends to
reaction and that substitution of Asn289 merely about 12 Å leading up to the active site and of enough
influences the rate of the reaction. Interestingly, neither length to model a C acyl group in it (Figure 4).
10
the Arg46(cid:198)Ala nor the Thr97(cid:198)Phe mutant exhibited Observation of electron density in this channel in both
decarboxylation activity in our hands. Although the the 1HZP and our Arg46Arg161(cid:198)Ala mutant
other mutants retained malonyl-AcpM decarboxylation structures suggests that this is a secondary acyl-binding
activity, they all catalysed the reaction at greatly channel.
reduced rates. Most interestingly, the
The orientation of Thr155 side chain is different
Arg46Arg161(cid:198)Ala mutant decarboxylated malonyl-
between the two double mutants. In the
AcpM more effectively than the Arg46(cid:198)Ala mutant.
Arg46Arg161(cid:198)Ala mutant, the orientation is fixed by
Presumably this effect is due to intragenic suppression
hydrogen bonding between the side chain hydroxyl and
5
backbone carbonyl oxygen of Leu152 so that the γ- DISCUSSION
methyl group of Thr155 faces away from channel 2. In
Previously we showed that mtFabH corresponds to the
the Trp42Arg161(cid:198)Ala mutant, the Thr155 side chain
E. coli KAS-III enzyme (FabH), although its substrate
γ-hydroxyl points towards the channel. Such a
specificity is different, preferring C -CoA rather than
difference in Thr155 side chain orientation would alter 12
acetyl-CoA (20). It was therefore proposed that mtFabH
the hydrophobicity of channel 2 that, in turn, could
represents the link between FAS-I and FAS-II in M.
change the acyl chain binding properties of this
tuberculosis by condensing the intermediate chain
channel.
length CoA-linked products of FAS-I with malonyl-
In the native and the mutant structures we determined, a AcpM, which following a complete cycle of FAS-II,
water molecule numbered 568 in Arg46Arg161(cid:198)Ala provides the acyl-AcpM primers for further extension,
mutant structure (we use the numbering in this mutant by KasA/B (20).
as other conserved waters not seen in the native and
In this study, we sought to analyze the influence of the
Trp42Arg161(cid:198)Ala mutant are discussed below) is
ACP partner on catalysis and to confirm the roles of
found at an average distance of 3.3 Å (Figure 5). This
several key amino acid residues suggested by the recent
water molecule is also hydrogen bonded to Nε2 of
determination of the mtFabH crystal structure. Analysis
His258 and backbone carbonyl oxygen of Tyr319 and
of mtFabH-catalysed malonate incorporation into β-
interacts with the active site Asn289. Qiu et al.
ketoacyl-ACP demonstrated an ACP-specific shift in
discussed the possibility of the role of a water molecule
acyl-CoA chain length preference. The optimal acyl-
in the deprotonation of Sγ of the active site Cys112 of
CoA primer determined with ecACP was C -CoA with
ecFabH (25). This water molecule is found in the 12
both C - and C -CoAs being efficiently extended
oxyanion hole formed by backbone amide nitrogens of 8 10
whereas longer acyl-CoAs represented poor substrates. D
Cys112 and the conserved Gly306. As the water ow
However when AcpM was used as the carrier, malonate n
molecule 568 is conserved in all the three structures we lo
incorporation continued to increase with acyl-CoA a
determined and interacts with all the three active site de
chain length to an apparent maximum with fatty acyl d
residues, we suggest this water might play a critical role primers ranging from C -CoA to C -CoA. The recent fro
in the deprotonation of Cys122. 14 20 m
determination of the crystal structure of a substrate h
Tbehtew esetnru catcutirvees sailtseo rerseivdeuaelse dC yas 12n2et wanodrk Hoisf2 5w8 ataenrds csuogmgpelsetxs thoatf famttytF aacbyHl -pCriyms1e2rs2 (cid:198)upA tola C 1w6-Citho A Cco12u-lCdo bAe ttp://ww
other parts of the protein. In all our FabH structures and accommodated within channel 2 (26,43). Our data with w.jb
the 1HZP structure, two water molecules are strictly ecACP, however, is consistent with C12-CoA being the c.o
conserved. One of them, numbered 352 in the optimal fatty acyl primer in the absence of AcpM. rg
Arg46Arg161(cid:198)Ala mutant structure is hydrogen However, since FAS-I products are distributed by/
g
bonded to backbone carbonyl oxygens of Ser291 and bimodally with the modal chain lengths occurring at ue
s
Ala293. Ser291 is equivalent to Ser276 of ecFabH that C16 and at C24 over the ranges C14-C18 and C20-C24 t on
has been implicated in deprotonation of the Cys122 respectively, C -CoA appears to have no physiological J
12 a
n
sulfydryl group (25). This water is at an average relevance. Clearly though, mtFabH can efficiently form u
a
distance of 3.9 Å from Sγ of Cys122 in these four acyl-enzyme intermediates with even longer primers in ry 8
structures. The other water molecule numbered 343 is the presence of AcpM and we suggest that these , 2
0
hydrogen bonded to Oε1 of Gln328 and backbone profiles of KAS-III activity demonstrate that AcpM- 19
carbonyl oxygen of Ala326. Water molecules numbered specific factors are required to promote frequent
436 and 437 are conserved between the 1HZP and acylation of mtFabH with available (C -CoA to C -
14 20
Arg46Arg161(cid:198)Ala mutant structures and complete the CoA) yet relatively unfavoured (without AcpM)
networks of waters that run from Ser291 carbonyl substrates in vivo.
oxygen, Cys122 Sγ and His258 Nδ1 to Gln328 Oε1 in
The crystal structures determined here and elsewhere
these structures (Figure 5). In the three structures we
show that residues Arg46, Arg228, Arg263 and Lys270
have determined, the side chain of Ser291 shows
contribute to the formation of an electropositive surface
rotational flexibility about the Cα-Cβ bond placing the
patch that would interact with the acidic AcpM. The
Oγ of the side chain at a distance of 3.7 Å, 2.4 Å and
existence of such an interaction surface on mtFabH also
3.7 Å from the Sγ of Cys122 in wild type,
explains why it is able to use ecACP in the in vitro
Trp42Arg161(cid:198)Ala and Arg46Arg161(cid:198)Ala mutant
reactions. Therefore, we suggest a similar mode of
structures respectively. Ser291 lies at the N-terminus of
binding for mtFabH/AcpM as has been suggested for
helix Cα3 suggesting that it has a lowered pKa. The
their E. coli counterparts (42). Sequence alignment of
side chain of Ser291 is also hydrogen bonded to
AcpM with ecACP revealed that the former bears a
Glu151. Glu151 can abstract a proton from Ser291 that
highly mobile 33-residue extended carboxy terminus
in turn can play a role in deprotonating Cys122. The
that may have an effect on protein:protein interactions
conservation of Ser291 in FabH from several organisms
or also interact with its bound acyl chain (44). Although
and its positioning is consistent with a possible role for
ACP is not necessary for transacylation of the enzyme,
this residue in catalysis.
there are several ways in which AcpM might influence
the apparent substrate selectivity of this enzyme. We
6
are currently developing experiments to determine the C acyl chains to the active site Cys residue (30). As
10
molecular basis of this modulation of fatty acyl primer the mtFabH His258(cid:198)Ala mutant retains some
preference and the identification of the crucial AcpM- transacylation activity, it is unlikely that this residue
specific factors involved. functions simply as a general base abstracting a proton
from Cys122 to promote acyl transfer as has been
We constructed several mutants, facilitating a
shown for chalcone synthase (45). Conserved waters
biochemical analysis of their individual contributions to
seen near the active site residues may also play a role in
KAS-III activity. Our analyses showed that Ala
the catalytic activity and a structural role to maintain
substitutions of catalytic triad residues (Cys122,
the appropriate active site geometry. The reduced
His258, Asn289) completely abrogated KAS-III
transacylation of the His258(cid:198)Ala mutant can be
activity confirming these as vital residues for the
explained by considering the role of His258 in
condensation reaction. The single substitutions of the
deprotonating the Cys122 sulfydryl group through a
acyl-CoA binding residues (Trp42(cid:198)Ala, Arg46(cid:198)Ala
water molecule (water 568) as follows: Nε2 of His258
and Arg161(cid:198)Ala) showed reduced KAS-III activity
can abstract a hydrogen from water 568 creating a
confirming a non-essential role for these residues in
hydroxyl anion, which in turn can deprotonate Cys122.
catalysis consistent with their predicted function in
We therefore propose this new role for His258 in the
binding of fatty acyl-CoA primers (26). The two double
transacylation reaction of mtFabH (Figure 6). However,
mutants (Trp42Arg161(cid:198)Ala and Arg46Arg161(cid:198)Ala),
as mutation of His258 does not eliminate activity, the
however, showed a more profound effect with almost
stabilization of the Cys122 thiolate ion and thus
complete loss of activity.
transacylation appears to be partially promoted via the
Next, we analysed mtFabH and our mutants in alpha-helix dipole effect as in ecFabH (24).
component part reactions to gain a clearer D
In ecFabH, active site residues His244 and Asn274 are ow
understanding of the roles of individual residues in n
both required for malonyl-ACP decarboxylation (24). lo
catalysis. When we measured approximate rates for a
Their side-chain nitrogen atoms form strong hydrogen de
protein transacylation, we found these to be relatively d
low compared to the rate of the condensation reaction. bonds with the thioester carbonyl oxygen, effectively fro
stabilizing the negative charge it gains during catalysis. m
However, the use of substrate-level concentrations of h
econnzydmenes ahtieorne adsoseasy sn owt haicllho win vsotrlivcet cmoumltpipalreis otunr ntoo vtehres Mddeeuccaatarrtbbioooxxnyy ollaaf ttiieooinnth era coatifcv ttiihtvyoi tsyeo fr ess(i2od4mu)ee.s aolmfT ohosetu re limmretidFnuaacbteeHdd ttp://ww
of the enzymes. Consequently, we view these mutants implicated several residues in promoting w.jb
observations with caution and suggest that they are c
efficient decarboxylation. As expected, the .o
merely illustrative of general trends rather than rg
providing quantitative comparisons of individual His258(cid:198)Ala and Asn289(cid:198)Ala substitutions almost by/
completely eliminated malonyl-AcpM decarboxylation. g
residue’s contribution to catalysis. The reaction u
Nearly all of the other mutant enzymes retain malonyl- es
appears to occur in two phases: an initial rapid phase, at AcpM decarboxylation activity but the reaction occurs t on
which substrate and enzyme are both plentiful, and a J
at significantly reduced rates similar to that of the an
phase at which both free enzyme and substrate are u
limited. With higher C -CoA concentrations, the rates Asn298(cid:198)Ala mutant. The reason for this reduced ary
10 activity is not known but it is possible that changes to 8
and extent of transacylation did increase although not , 2
the active site geometry brought about by these 0
proportionately (data not shown) and still did not tend 19
substitutions could significantly reduce the reaction
towards either complete or 50 % saturation, both of
rate. Surprisingly, Arg46(cid:198)Ala greatly reduces the
which could be coherently reconciled with a
decarboxylation activity of mtFabH. Arg36 of ecFabH
mechanistic model. Together these observations may
was predicted to interact with Ser36 of ecACP (42) to
suggest that another factor is required for efficient
which a phosphopantetheine arm that carries acyl
transacylation, possibly an interaction with malonyl-
chains is attached. In the absence of experimentally-
AcpM at the opposite monomer. The different degrees
derived structural data regarding the mode of binding of
of binding observed with each of our mutants probably
malonyl-AcpM by mtFabH, it remains possible that this
reflects individual changes in substrate ‘affinity’ that
residue plays a major role in the binding of AcpM,
influences the rate of the initial rapid transacylation
possibly interacting with the Ser41-phosphopantetheine
phase in these assays.
moiety of AcpM (see supplementary data, Figure S2).
Both of the mutants substituted at catalytic triad The possibility that this single residue plays a role in
residues (His258(cid:198)Ala and Asn289(cid:198)Ala residues) the binding of both AcpM and the acyl-CoA primer,
were transacylated to a reduced extent compared to albeit at different times, requires further investigation.
mtFabH. These mutations had a more profound effect Incorporating interactions with such a residue may
on acyl transfer than did the individual substitutions in improve the efficacy of novel inhibitors of these
residues previously implicated in acyl-CoA binding. enzymes. Interestingly, some recovery of malonyl-
The molecular basis for this is less clear. These two AcpM decarboxylation activity relative to Arg46(cid:198)Ala
polar residues may influence the binding of the CoA is seen in the Arg46Arg161(cid:198)Ala mutant, suggesting
moiety or aid the transfer of the acyl chain to the active our alteration of Arg161 is acting as an intragenic
site Cys residue. Interestingly, Ala substitution of E. suppressor mutant of the Arg46(cid:198)Ala substitution and
coli FabB at His289 also resulted in reduced transfer of possibly indicating the importance of local charge
7
distribution to efficient decarboxylation. These two 13. Smith, S., Witkowski, A., and Joshi, A. K. (2003) Prog
arginine residues are located close to the opening of Lipid Res 42, 289-317
channel 1; Arg46 neighbors Trp42 on a short helix with 14. Lu, Y. J., Zhang, Y. M., and Rock, C. O. (2004) Biochem
Cell Biol 82, 145-155
Arg161 opposite forming part of a short loop. As a
15. Bloch, K. (1977) Adv Enzymol Relat Areas Mol Biol 45,
similar effect was not observed in our transacylation
1-84
study, it would appear that this effect is specific for
16. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher,
interactions with AcpM.
C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S.,
Barry, C. E., 3rd, Tekaia, F., Badcock, K., Basham, D.,
Crystal structures of wild type, Arg46Arg161(cid:198)Ala and
Brown, D., Chillingworth, T., Connor, R., Davies, R.,
Trp42Arg161(cid:198)Ala mutants of mtFabH have been
Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd,
determined and found to be almost identical. Acyl chain
S., Hornsby, T., Jagels, K., and Barrell, B. G. (1998)
binding in the channel 2 in the Arg46Arg161(cid:198)Ala Nature 393, 537-544
mutant is consistent with the higher transacylation 17. Mdluli, K., Slayden, R. A., Zhu, Y., Ramaswamy, S., Pan,
activity of this mutant compared with that of the X., Mead, D., Crane, D. D., Musser, J. M., and Barry, C.
Trp42Arg161(cid:198)Ala mutant. The orientation of Thr155 E., 3rd. (1998) Science 280, 1607-1610
side chain is different between these two mutants. In 18. Peterson, D. O., and Bloch, K. (1977) J Biol Chem 252,
the Arg46Arg161(cid:198)Ala mutant, the γ-methyl group of 5735-5739
19. Slayden, R. A., Lee, R. E., Armour, J. W., Cooper, A. M.,
Thr155 faces away from channel 2 but in the
Orme, I. M., Brennan, P. J., and Besra, G. S. (1996)
Trp42Arg161(cid:198)Ala mutant the γ-hydroxyl points
Antimicrob Agents Chemother 40, 2813-2819
towards the channel. Such a difference would alter the
20. Choi, K. H., Kremer, L., Besra, G. S., and Rock, C. O.
hydrophobicity of channel 2, which, in turn, could
(2000) J Biol Chem 275, 28201-28207
affect the acyl chain binding properties of this channel. 21. Kremer, L., Douglas, J. D., Baulard, A. R., Morehouse, D
C., Guy, M. R., Alland, D., Dover, L. G., Lakey, J. H., ow
These studies have increased our understanding of the Jacobs, W. R., Jr., Brennan, P. J., Minnikin, D. E., and nlo
roles played by key mtFabH residues in catalysis and a
Besra, G. S. (2000) J Biol Chem 275, 16857-16864 d
e
htearvmes leoafd tthoe thsueb sdtervateel opsmpeecnift icoitfy nedwis phlayypeodt hebsye s thine 22. 5H8e1a-t5h9, 6R . J., and Rock, C. O. (2002) Nat Prod Rep 19, d from
enzyme; both factors which should facilitate further 23. Schaeffer, M. L., Agnihotri, G., Volker, C., Kallender, H., h
research into novel anti-tubercular agents. 2B7r6en, n4a7n0,2 P9.- 4J.7, 0a3n7d Lonsdale, J. T. (2001) J Biol Chem ttp://w
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24. Davies, C., Heath, R. J., White, S. W., and Rock, C. O. w
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46 Kabsch, W. (1976) Acta Cryst A32, 922-923

D
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fro
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h
ttp
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.jb
c
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rg
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1
9
9
Table I. Crystallographic data and refinement statistics
mtFabH Trp42Arg161(cid:198)Ala Arg46Arg161(cid:198)Ala
Data collection :
Space group C2 P2(1) C2
Cell dimensions (a, b, c 103.31, 80.9, 96.33, 64.17, 56.02, 88.19, 103.45, 81.41, 97.28,
Å, β°) 106.08° 90.57° 106.91°
Resolution (Å) 30-2.65 50-2.5 50-2
Completeness % (last 97 (97.5) 96.5 (98.6) 99.7 (97.8)
shell)
I/σ(I) (last shell) 15.6 (2.8) 13.1 (2.6) 26.4 (2.4)
No. of total reflections 51681 67479 213586
No. of unique reflections 21140 21086 50640
R % (last shell) 6.8 (37.8) 8.4 (36.2) 5.1 (52.8)
sym

Refinement :
D
Resolution ( Å) 30-2.7 40-2.5 30-2 o
w
n
No. of reflections in test lo
a
set 1924 1980 2485 de
d
No. of reflections in fro
m
working set 19658 18183 46972 h
ttp
No. of protein molecules ://w
w
per asu 2 2 2 w
.jb
No. of protein atoms in c.o
asu 4828 4856 4862 rg
b/
y
No. of water molecules in g
u
asu 78 100 256 es
t o
n
R-factor (%) 21.2 22.5 24.8 J
a
n
u
Rfree (%) 26.5 27.9 27.8 ary
8
, 2
0
1
RMS deviations 9
Bond lengths (Å) 0.008 0.008 0.005
Bond angles (degrees) 1.5 1.32 1.18

Average B-factors (Å2)
Main chain 39.1 41.3 33.2
Side chain and waters 42.1 43.4 34.9
RMS B main chain 1.02 0.96 1.02
RMS B side chain 1.48 1.45 1.36

10