Expanding metabolism for biosynthesis
of nonnatural alcohols
Kechun Zhanga, Michael R. Sawayab,c, David S. Eisenbergb,c, and James C. Liaoa,c,1
Departments ofaChemical and Biomolecular Engineering and Chemistry andbBiochemistry andcInstitute for Genomics and Proteomics, University of
California, Los Angeles, CA 90095
Edited by Frances H. Arnold, California Institute of Technology, Pasadena, CA, and approved November 4, 2008 (received for review July 24, 2008)
Nature uses a limited set of metabolites to perform all of the
biochemical reactions. To increase the metabolic capabilities of
biological systems, we have expanded the natural metabolic net-
work, using a nonnatural metabolic engineering approach. The
branched-chain amino acid pathways are extended to produce
abiotic longer chain keto acids and alcohols by engineering the
chain elongation activity of 2-isopropylmalate synthase and alter-
ing the substrate specificity of downstream enzymes through
rational protein design. When introduced into Escherichia coli, this
nonnatural biosynthetic pathway produces various long-chain al-
cohols with carbon number ranging from 5 to 8. In particular, we
demonstrate the feasibility of this approach by optimizing the
biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This
work demonstrates an approach to build artificial metabolism
beyond the natural metabolic network. Nonnatural metabolites
of living systems.
metabolic engineering ? protein engineering ? chain elongation ?
long chain alcohols
blocks for biosynthesis. These chemicals support the biological
functions of all organisms. So far, construction of artificial
biological systems (1–5) is limited by the existing metabolic
capabilities. By supplying living cells with chemically synthesized
nonnatural amino acids (6, 7) and sugars (8) as new building
blocks, it is possible to introduce novel physical and chemical
properties into biological entities. These efforts raise an inter-
esting question: Can we rewire metabolism in a bottom-up
fashion to produce nonnatural metabolites from simple carbon
source? If so, such engineered artificial metabolism should be
able to expand the chemical repertoire that living systems can
a strategy to produce 7-(C7) to 9-carbon (C9) 2-keto acids, which
can lead to useful nonnatural alcohols (C6–C8).
Aliphatic alcohols with carbon chain length ?5 (C ? 5) are
attractive biofuel targets because they have higher energy den-
sity, and lower water solubility [1-pentanol 23 g/L, 1-hexanol 6.2
g/L, 1-heptanol 1.2 g/L (9)] that could facilitate postproduction
purification from culture medium through an aqueous/organic
2-phase separation process. The only well-characterized mech-
anism for aliphatic alcohol production is through the Ehrlich
pathway (10), which converts branched-chain amino acids into
alcohols. The carbon number (up to 5) of the alcohols derived
from this type of pathway is limited by the carbon number in the
branched chain amino acid pathways (11). To overcome this
limitation, existing metabolic networks need to be expanded.
This is a daunting task because a metabolic pathway usually
involves the collective function of multiple enzymes, which have
to be engineered by rational design (12) or directed evolution
(13, 14) to perform nonnatural activities.
Acetyl-CoA is a common chemical unit for carbon chain
elongation, such as reactions in tricarboxylic acid cycle, glyoxy-
late cycle, mevalonate pathway, and leucine biosynthesis (15). To
ature uses a limited set of metabolites such as organic acids,
amino acids, nucleotides, lipids and sugars as building
explore the possibility of using acetyl-CoA related chemistry to
produce C6 alcohol, we have engineered a nonnatural metabolic
pathway (Fig. 1B, shaded region) into E. coli. First, we used the
existing metabolic capability of E. coli to synthesize (S)-2-keto-
3-methylvalerate, the 2-keto acid precursor of amino acid L-
isoleucine. The chemical structure of 2-keto-3-methylvalerate is
Author contributions: K.Z., M.R.S., and J.C.L. designed research; K.Z. performed research;
K.Z. and J.C.L. analyzed data; and K.Z., M.R.S., D.S.E., and J.C.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Los Angeles, CA 90095. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
Il C Il D IlvC, IlvD
(ADH6). (B) Schematic representation of the biosynthetic pathway of 3-methyl-
1-pentanol. The engineered nonnatural metabolic pathway is shaded in laven-
der. Similar to 2-ketoisovalerate, 2-keto-3-methylvalerate can have one more
carbon added to its side chain by the leucine biosynthesis enzymes. (C) Synthetic
operons for gene expression. Overexpression of ThrABC, TdcB and IlvGMCD
drives the carbon flux toward 2-keto-3-methylvalerate.
Pathway design. (A) Conversion of 2-keto acids to alcohols by a broad-
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vol. 105 ?
no. 52 ?
very similar to 2-ketoisovalerate (the 2-keto acid precursor of
amino acid L-valine), containing only one more methyl group on
the side chain. Because 2-ketoisovalerate is converted to 2-ke-
toisocaproate through a 3-step chain elongation cycle by 2-iso-
propylmalate synthase (LeuA), isopropylmalate isomerase com-
plex (LeuC, LeuD) and 3-isopropylmalate dehydrogenase
(LeuB), we reasoned that LeuA, LeuB, LeuC and LeuD may be
promiscuous enough to allow 2-keto-3-methylvalerate go
through the same elongation cycle and produce a novel com-
pound 2-keto-4-methylhexanoate. Analogous to the Ehrlich
that 2-keto-4-methylhexanoate could be converted to the cor-
responding aldehyde and then to a 6-carbon alcohol, (S)-3-
methyl-1-pentanol, by the broad-substrate-range 2-ketoisovaler-
ate decarboxylase (KIVD) from Lactococcus lactis (16) and
alcohol dehydrogenase VI (ADH6) from Saccharomyces
Results and Discussion
Construction of a Nonnatural Metabolic Pathway for Biosynthesis of
(S)-3-Methyl-1-Pentanol. We constructed 3 synthetic operons (Fig.
1C) under the control of the PLlacO1 promoter: The first operon
is composed of 3 genes on a low copy plasmid in the transcrip-
tional order thrA-thrB-thrC; the second operon is composed of 5
genes on a medium copy plasmid in the transcriptional order
tdcB-ilvG-ilvM-ilvC-ilvD, and the third operon is composed of 6
genes on a high copy plasmid in the transcriptional order
leuA-leuB-leuC-leuD-KIVD-ADH6 (and a control operon with-
out leuABCD). Except for KIVD, ADH6 and ThrA (G433R
mutant (18) insensitive to threonine feedback inhibition), all
other genes encode wild-type E. coli enzymes. As a result of
overexpressing these 14 genes in a modified threonine-
hyperproduction strain (ATCC98082, ?ilvE, ?tyrB), 6.5 mg/L of
(S)-3-methyl-1-pentanol was produced from 20 g/L glucose
(Table 1, column 4), whereas a leucine-feedback insensitive
G462D mutant (19) LeuA produced 40.8 mg/L of C6 alcohol
(Table 1, column 5). In contrast, without overexpression of
LeuABCD, no C6 alcohol production was detected (Table 1,
Structure-Based Redesign of KIVD. Because KIVD and ADH6 are
promiscuous enzymes, they can also convert other intracellular
2-keto acids into alcohols (Fig. 1B, Table 1). To reduce the
formation of byproducts and drive the carbon flux toward the
target C6 alcohol, we examined the effect of engineering KIVD
with higher selectivity toward 2-keto-4-methylhexanoate. The
protein sequence alignment shows that KIVD has 40% and 31%
sequence identities with Enterobacter cloacae indolepyruvate
decarboxylase IPDC (20) and Zymomonas mobilis pyruvate
decarboxylase ZmPDC (21) respectively. A homology model for
the substrate-binding region of KIVD and IPDC was built based
on the crystal structures of ZmPDC (PDB: 1ZPD). As can be
seen from Fig. 2, 4 residues, Ser-286, Phe-381, Val-461, and Met
538, in combination with cofactor thiamine diphosphate
(ThDP), delineate the keto-acid binding pocket of KIVD. No-
chains, Tyr-290, Trp-392, Ile-472, and Trp-551; and those of
IPDC have smaller ones, Thr-290, Ala-387, Val-467 and Leu-
542. These differences can explain the substrate spectrum of
related amino acids might be able to change substrate specificity.
A ZmPDC I472A variant was shown to be more active on
longer-chain keto acids other than pyruvate (22). The corre-
sponding residue of KIVD, V461, was thus mutated to alanine.
Compared with the wild-type KIVD, the V461A mutant pro-
duced 3 times more 3-methyl-1-pentanol (Table 1, column 6).
Further optimization was performed by mutating either F381 or
M538 to smaller hydrophobic side chains such as leucine or
alanine (Table 1). The F381L/V461A mutant was the best variant
obtained and produced 384.3 mg/L of 3-methyl-1-pentanol.
Both wild-type and F381L/V461A KIVD were added an
N-terminal 6xHis-tag, overexpressed and purified through Ni-
NTA columns. The kinetic parameters for activation of 2-ke-
toisovalerate (cognate substrate) and 2-keto-4-methylhexanoate
Table 1. Production profile of alcohols from the designed pathway, with different KIVD mutants
Alcohol titer (mg/L)
41.1±4.194.6±11.5 213.2±12.3 132.7±14.327.3±5.1100.7±18.043.3±12.9 83.3±6.2
1179.1±76.5936.2±42.781.8±19.149.6±12.9 5.3±2.937.3±8.116.1±3.3 8.0±1.1
ND17.8±0.9 493.2±31.5371.4±14.6192.1±7.7432.1±52.0219.3±51.7381. 7±36.3
54.1±5.563.4±14.8205.2±9.4 264.5±9.9142.9±10.5246.2±38.0 122.8±33.668.0±6.7
NDND494.1±22.9503.9±4.6 750.5±52.9 556.6±86.8482.9±111.9444.6±35.5
NDND NDND17.4±0.3 ND18.5±0.97.3±0.4
Note that the V461A/F381L mutant gives the highest titer of 3-methyl-1-pentanol. E. coli cultures were grown in M9 medium with 20 g/L glucose plus 0.1 mM
IPTG at 30 °C for 40 h. These products were identified by GC-MS and quantified by GC-FID. ND, not detectable.
www.pnas.org?cgi?doi?10.1073?pnas.0807157106 Zhang et al.
(nonnatural substrate) were determined using a coupled enzy-
matic assay (22). Compared with the wild-type KIVD, for the
smaller substrate, 2-ketoisovalerate, F381L/V461A KIVD has a
mM versus 2.2 mM); for 2-keto-4-methylhexanoate, F381L/
V461A KIVD has a comparable kcat(3.0 s?1versus 10.8 s?1) and
a slightly higher Km (0.22 mM versus 0.14 mM). Thus, the
specificity constant kcat/Kmof F381L/V461A KIVD toward 2-ke-
to-4-methylhexanoate is 40-fold higher than that toward 2-ke-
toisovalerate. In comparison, the specificity constant kcat/Kmof
wild-type KIVD toward 2-keto-4-methylhexanoate is only 4-fold
higher than that toward 2-ketoisovalerate (Table 2). Such a
change in KIVD specificity distinguishably affects the distribu-
tion profile of alcohol products (more long-chain alcohols and
less short- chain alcohols).
Enlarging the Binding Pocket of LeuA. Besides KIVD, the other key
enzyme determining the carbon flux toward 3-methyl-1-
pentanol production is LeuA. LeuA catalyzes the condensation
of acetyl-CoA with 2-keto-3-methylvalerate, which is the first
step of the expanded metabolic pathway (Fig. 1B). LeuA also
competes with KIVD for substrate 2-keto-3-methylvalerate, and
thus reduces the formation of side product 2-methyl-1-butanol.
Engineering KIVD with higher activity toward 2-keto-3-
methylvalerate should help increase 3-methyl-1-pentanol pro-
duction. As inferred from the crystal structure of Mycobacterium
tuberculosis LeuA (23), residues His-167, Ser-216 and Asn-250
are within a radius of 4 Å of the ?-methyl group of bound
2-ketoisovalerate (Fig. 3A). Nonnatural substrate (S)-2-keto-3-
methylvalerate contains one more methyl group that would
cause steric hinderance with Ser-216, which could be relieved by
mutating serine to the smallest amino acid glycine. Multiple
protein sequence alignment shows that E. coli LeuA shares 92%
and only 21% sequence identity with Salmonella typhimurium
LeuA and M. tuberculosis LeuA respectively. Fortunately, the
binding pocket is well conserved and the corresponding residues
of E. coli LeuA are His-97, Ser-139 and Asn-167 (Fig. 3B). The
G462D/S139G mutant LeuA was cloned and produced 793.5
by G462D LeuA.
Enzymatic assay indicates that G462D LeuA has an extremely
low kcat (0.018 s?1) for (S)-2-keto-3-methylvalerate, which is
333-fold less than that for 2-ketoisovalerate (6.0 s?1). Because
G462D LeuA has a comparable Kmfor both substrates (55 ?M
versus 182 ?M), the low kcat may be why a previous report
showed that 2-keto-3-methylvalerate is a strong inhibitor of
LeuA (24). However, the S139G mutation increases the kcat
7-fold for (S)-2-keto-3-methylvalerate to 0.12 s?1(Table 4).
Additional mutations were then performed on His-97 and
Asn-167. Even though better mutant was not found for produc-
tion of 3-methyl-1-pentanol, interestingly, the G462D/S139G/
N167A triple mutant produced 51.9 mg/L 4-methyl-1-hexanol
(C7), and the G462D/S139G/H97A/N167A quadruple mutant
(green) and the corresponding homology model of Enterobacter cloacae
indolepyruvate decarboxylase IPDC (cyan) and KIVD (purple), using ZmPDC as
the template. The multiple sequence alignment was performed with Clust-
V461, and M538 of KIVD were mutated to smaller hydrophobic residues such
as alanine and leucine to allow the enzyme accept substrates larger than
Table 2. Kinetic parameters of wild type and mutant KIVD
Wild type V461A/F381L
2-Ketoisovalerate2.2± 0.938.3± 9.817 7.7± 1.8 2.7± 0.60.35
0.14± 0.0110.8± 0.3 77 0.22± 0.02 3.0± 0.1 14
terium tuberculosis LeuA (PDB: 1SR9) complexed with its natural substrate
2-ketoisovalerate (KIV, green). (S)-2-keto-3-methylvalerate has one more
(red sphere), His-167, and Asn-250. (B) Multiple sequence alignment of M.
tuberculosis, E. coli, and Salmonella typhimurium LeuA. The binding pocket is
conserved, and the corresponding residues of E. coli LeuA are His-97, Ser-139,
and Asn-167. These residues were subjected to site-specific mutagenesis.
Residues in the active site of LeuA. (A) Binding pocket of Mycobac-
Zhang et al.
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produced 57.3 mg/L 4-methyl-1-hexanol (C7) and 22.0 mg/L
Biosynthesis of a Repertoire of Nonnatural Alcohols. Because the
engineered LeuA has larger binding pockets, the chain elongation
activities may continue several more rounds by LeuA on the 2-keto
acids produced from the LeuABCD or other pathways (Fig. 4A).
For example, 2-ketobutyrate can be converted to 2-ketovalerate,
then to 2-ketocaproate, and finally to 2-ketoheptanoate by
LeuABCD. In parallel, 2-keto-3-methylvalerate can be converted
to 2-keto-4-methylhexanoate, then to 2-keto-5-methylhepatanoate
of F381L/V461A KIVD. Upon decarboxylation, the corresponding
aldehydes are reduced to the corresponding alcohols by ADH6.
Indeed, we observed accumulation of 5 other nonnatural alcohols:
1-pentanol, 1-hexanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol
and 5-methyl-1-heptanol (Tables 1 and 3). The anteiso-methyl-
branched alcohols are all derived from the same chiral precursor,
(S)-2-keto-3-methylvalerate. The S-configuration of the stereo-
genic center in these alcohols remains unchanged during biosyn-
thesis as confirmed by chiral GC analysis of (S)-2-methyl-1-butanol
[see supporting information (SI)] and (S)-3-methyl-1-pentanol
synthons for chemical synthesis (25).
In this work, we have shown that by combining protein engi-
neering and metabolic engineering approaches, it is possible to
expand the intermediary metabolism of E. coli to produce
various C5 to C8 alcohols that are not readily produced by
Table 4. Kinetic parameters of wild type and mutant LeuA
2-Ketoisovalerate 182± 26.0± 0.3
33104± 5 2.1± 0.120
55± 60.018± 0.001
0.33144± 130.12± 0.020.83
Table 3. Alcohol production with different LeuA mutants
Alcohol titer (mg/L)
49. 6±2.270.0±9.0 155.2±12.3 165.1±18.6208.1±8.3 230.4±39.1
178.5±5.5174.1±13.1 25.2±4.230.6±2.6 28.6±2.417.9±6.3
204.7±16.5169.8±36.529.9±4.417.3±0.514. 2±1.3 ND
793.5±46.5685.7±16.0337.4±41.0 288.1±32.5119.1±6.0 290.6±34.1
37.4±2.8 38.4±8.316.6±0.916.5±1.4 NDND
NDND ND51.9±9.3 ND57.3±7.8
NDND ND NDND22.0±2.5
Note that the G462D/S139G mutant gives the highest titer of 3-methyl-1-pentanol). E. coli cultures were grown in M9 medium with 20 g/L glucose plus 0.1
mM IPTG at 30 °C for 40 h. These products were identified by GC-MS and quantified by GC-FID. ND, not detectable.
www.pnas.org?cgi?doi?10.1073?pnas.0807157106Zhang et al.
microorganisms. Because of their specific physical and chemical
properties, these long chain alcohols could be good candidates
as biofuels or renewable chemical reagents. For practical appli-
cations, further metabolic engineering (26) and enzyme engi-
neering (27) will be needed to increase the production yield and
rate of these compounds. Because the 2-keto acid precursors of
alcohols can be converted to amino acids by aminotransferases,
we hereby also provide a biosynthetic way, instead of traditional
organic synthesis, to expand the repertoire of nonnatural amino
acids that have recently found broad applications (6, 28).
Materials and Methods
Vector Construction. All cloning procedures (see SI for cloning scheme) were
carried out in the E. coli strain XL10-gold (Stratagene). Oligos were synthe-
sized by Operon Biotechnologies (see SI for sequence details). PCRs were
performed with KOD polymerase (Novagen).
Fermentation Procedure. The aminotransferase genes, ilvE and tyrB, of a
threonine-hyperproduction E. coli strain ATCC98082 were inactivated by P1
transduction (29). This modified strain was transformed with pZS?thrO,
pZAlac?tdcBilvGMCD and pZE?LeuABCDKA6 for alcohol production. Over-
night cultures incubated in LB medium were diluted 100-fold into 5 mL of M9
medium supplemented with 1? trace metal mix A5 (11), 0.5% yeast extract
(ampicillin 100 mg/L, spectinomycin 25 mg/L, kanamycin 25 mg/L). Cells were
mM isopropyl-b-D-thiogalactoside (IPTG). Cultures were then transferred to a
30 °C shaker (250 rpm) and incubated for 40 h.
GC-MS Analysis. The GC-MS system is composed of model 6890N network GC
system (Agilent Technologies), a model 7883B injector and autosampler (Agi-
Technologies). Samples were separated through a DB-5ms capillary column
(30 m, 0.25-mm internal diameter, 0.25-?m film thickness; Agilent Technolo-
gies) with helium (1 mL?min?1) as the carrier gas. Alcohols extracted by 200 ?L
equipped with flame ionization detector. The system is composed of a model
5890A gas chromatograph (Hewlett Packard) and a model 7673A automatic
injector, sampler and controller (Hewlett Packard). Samples were separated
through A DB-FFAP capillary column (30 m, 0.32-mm internal diameter,
0.25-?m film thickness; Agilent Technologies). GC oven temperature was
initiallyplacedat40 °Cfor2min,increasedwithagradientof5 °Cmin?1until
45 °C, and held for 4 min. Then it was increased with a gradient 15 °C min?1
until 230 °C and held for 4 min. Helium was used as the carrier gas. The
temperature of injector and detector was set at 225 °C. Alcohol standards
were purchased from either Sigma–Aldrich or TCI America.
For chiral GC analysis, samples were separated through a HP-CHIRAL 20ß
column (30 m, 0.32-mm internal diameter, 0.25-?m film thickness; Agilent
Technologies). The racemic mixture of 3-methyl-1-pentanol could not be
directly resolved. However, after reaction with N-Methyl-N-[tert-butyldim-
ethyl-silyl]trifluoroacetimide (Pierce), the conjugated product could be re-
solved into 2 peaks. GC oven temperature was initially placed at 50 °C for 4
min, increased with a gradient of 10 °C min?1until 90 °C, and held for 2 min.
Andthenitwasincreasedwithagradient2 °Cmin?1until130 °Candheldfor
of injector and detector was set at 225 °C.
ProteinExpressionandPurification.Both gene fragments encoding wild-type
and F381L/V461A KIVD were amplified from plasmid pZE?LeuABCDKA6,
using primers hiskivd?tevfwd and hiskivd?bamrev. After digestion with
BamHI, the gene fragments were inserted into expression plasmid pQE9
(Qiagen) to yield pQE?hiskivd?wt and pQE?hiskivd?FL. The ADH6 gene
fragment was amplified from yeast genomic DNA, using primers
hisadh?tevfwd and hisadh?bamrev, digested with BamHI and inserted into
pQE9 to generate pQE?hisadh6. Similarly, genes encoding G462D and
G462D/S139G LeuA were amplified from plasmid pZE?LeuABCDKA6, using
primers hisleua?tevfwd and hisleua?bamrev. After digestion with BamHI,
the PCR products were ligated into pQE9 to create pQE?hisleua?GD and
pQE?hisleua?GS. The resulting expression plasmids pQE?hiskivd?wt,
pQE?hiskivd?FL, pQE?hisadh6, pQE?hisleua?GD and pQE?hisleua?GS were
transformed into E. coli strain BL21(DE3) harboring pREP4 (Qiagen). Cells
were inoculated from an overnight preculture at 1/100 dilution and grown
in 200 mL of 2XYT rich medium containing 50 mg/L ampicillin and 25 mg/L
kanamycin. At an OD600of 0.6, recombinant proteins were expressed by
S-isomer as confirmed by chiral GC analysis after MTBSTFA (N-Methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide) derivatization.
Structures of biosynthesized alcohols. (A) Nonnatural alcohols produced and their corresponding metabolic pathways. (B) 3-methyl-1-pentanol is
Zhang et al.
December 30, 2008 ?
vol. 105 ?
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induction of the cell cultures with 0.1 mM IPTG, followed by incubation at
30 °C overnight. Cell pellets were lysed by sonication in a buffer containing
250 mM NaCl, 2 mM DTT, 5 mM imidazole and 50 mM Tris pH 9.0. By
applying a stepwise gradient of imidazole (up to 250 mM), enzymes were
purified from crude cell lysates through Ni-NTA column chromatography.
The fractions of highest purity were pooled and buffer-exchanged using
Amicon Ultra centrifugal filters (Millipore). Storage buffer 1 [50 ?M Tris
buffer (pH 8.0), 1 mM MgSO4, 20% glycerol] was used for LeuA and ADH6,
and storage buffer 2 [50 ?M Tris buffer (pH 8.0), 1 mM MgSO4, 0.2 mM
ThDP, 20% glycerol] was used for KIVD. The concentrated protein solutions
were aliquoted (100 ?L) into PCR tubes and flash frozen at ?80 °C for long
Enzymatic Assay of KIVD. Substrate 2-ketoisovalerate (KIV) was purchased
from Sigma–Aldrich, and (S)-2-keto-4-methylhexanoate (KHV) was custom
synthesized by AsisChem Inc. Protein concentration was determined by mea-
suring UV absorbance at 280 nm. The decarboxylation activity of KIVD was
used to reduce aldehyde into alcohol, and concomitantly, cofactor NADPH
was oxidized to NADP?. The assay mixture contained 0.2 mM NADPH, 0.1 ?M
ADH6 and 0.1–20 mM 2-keto acids in assay buffer (50 mM potassium phos-
phate buffer, pH 6.8, 1 mM MgSO4, 0.5 mM ThDP) with a total volume of 0.2
mL. The reactions were started by adding 2 ?L of KIVD (final concentrations:
for KIV, 20 nM wild-type KIVD, 200 nM F381L/V461A KIVD; for KHV, 50 nM
both), and the consumption of NADPH was monitored at 340 nm (extinction
coefficient, 6.22 mM?1?cm?1). Kinetic parameters (kcatand Km) were deter-
MgCl2, 1 mM acetyl-CoA and 100 mM Tris (pH 8.0) with a total volume of 100
?L. G462D or G462D/S139G LeuA (each 100 nM) was reacted with 2-ke-
while 4 ?M G462D or 1.5 ?M G462D/S139G LeuA was reacted with (S)-2-keto-
3-methylvalerate in a concentration range from 50 ?M to 2 mM for 30 min at
30 °C. The reactions were stopped by adding 0.3 mL of ethanol. Then 0.2 mL
buffer (pH 8.0) was added, and the yellow color product was measured at 412
nm. The values obtained were corrected for unspecific hydrolysis by subtract-
ing the absorbance of controlled samples without addition of 2-keto acids. A
molar extinction coefficient of 13,600 M?1?cm?1was used in the final calcu-
ACKNOWLEDGMENTS. This work was supported in part by the University of
California, Los Angeles Department of Energy Institute for Genomics and
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