A R T I C L E S
Vedha-Peters et al.
restriction enzymes and transformed into electrocompetent E. coli
(TOP10 cell line).
such as methylene blue or 2,6-dichloroindophenol in the
oxidative deamination direction. D-Amino acid synthesis via the
reductive amination reaction direction was not described. In
addition, most of these enzymes are membrane bound, and it
would therefore be difficult to obtain high yields of soluble and
active enzyme from a suitable expression host such as E. coli.
Misono and co-workers have also described the isolation of an
NADP+ dependent D-theronine deydrogenase from Pseudomo-
nas cruciViae.18 However, this enzyme catalyzes the oxidation
of the â-hydroxyl and not the oxidation/reduction of the R-amine
and is different from the enzyme described in this work.
In this work we report the creation of the first broad substrate
range, nicotinamide cofactor dependent, and highly D-enantio-
selective amino acid dehydrogenase. This enzyme was created
by performing directed evolution on the starting enzyme meso-
2,6-D-diaminopimelic acid dehydrogenase (DAPDH) using both
rational and random mutagenesis.
B. Synthesis of Mutant Library. Mutant libraries of the DAPDH
gene were created using error prone PCR techniques. To accomplish
this, 50 pmol of each primer that anneals to the pTrcHis2A vector
flanking the DAPDH gene insert (forward: 5′-GAGGTATATATTAT-
TGTATCG-3′ and reverse: 5′-GATGATGATGATGGTCGACGG-3′)
and 0.1 µg of DAPDH/pTrcHis2A plasmid DNA template were added
to 1.5 mM MnCl2, 5.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dGTP, 1.0
mM dCTP, 1.0 mM dTTP, 1X Taq polymerase PCR buffer and 2.5
units of Taq polymerase. The thermal cycling parameters were 94 °C
for 2 min (1 cycle), 94 °C for 45 s, 52 °C for 45 s, 72 °C for 90 s (30
cycles), and 72 °C for 10 min (1 cycle). The PCR products were
digested with DpnI to remove template DNA. The PCR products were
gel purified and digested with NcoI and XhoI. The digested products
were then gel-purified, and the DAPDH fragments were ligated into
the similarly digested vector pTrcHis2A. Ligation mixtures were
transformed into E. coli TOP10 cells by electroporation and selected
on LB medium supplemented with 100 µg/mL ampicillin.
For site-saturation mutagenesis a similar procedure was used. The
PCR reaction included the same forward and reverse primers used to
produce the error prone PCR library, internal oligos containing an NNN
sequence at the site to be saturated mutagenized with a 15 bp overlap
on both sides of the NNN, 0.2 mM dNTP, and 5.5 mM MgCl2. The
thermal cycling parameters were identical to those used in the error
prone PCR reaction, as was the PCR product purification. The multiple
PCR products (0.2 µg/fragment) from this reaction were combined with
a second PCR reaction containing 50 pmol of the same forward and
reverse primers, 0.2 mM dNTP, and 5.5 mM MgCl2. The thermocycling
parameters were as follows: 94 °C for 2 min (1 cycle), 94 °C for 45 s,
48 °C for 45 s, (10 cycles), 94 °C for 45 s, 52 °C for 45 s, 72 °C for
90 s (30 cycles), and 72 °C for 10 min (1 cycle). The final PCR product
was ligated and transformed as described above.
Materials and Methods
Materials. NADP+ and NADPH were obtained from BioCatalytics,
Inc. (Pasadena, CA). D-Amino acids were obtained from Sigma-Aldrich
Chemical Co. (St. Louis, MO) or Peptech Corp. (Burlington, MA).
2-Keto acids were purchased from Sigma-Aldrich or, if unavailable
commercially, were prepared by the addition of the corresponding
aldehyde to hydantoin followed by saponification as described previ-
ously.19 Nitro Blue Tetrazolium, phenazine methosulfate, and Triton
X-100 were obtained from Sigma-Aldrich. FMOC-Cl (used to
derivatize amino acids for UV detection) was purchased from Pierce
Biotechnology, Inc. (Rockford, IL). All other chemicals were purchased
from Sigma-Aldrich or similar sources and were of reagent grade or
higher.
C. Screening of Mutant Library. Individual colonies were picked
using an AutoGenesys robotic colony picker (AutoGen, Framingham,
MA), into 384-well microtiter plates containing Terrific Broth (TB)
media with 100 µg/mL ampilicillin and grown 16 h at 37 °C. After
growth the master plates were replicated into 384-well plates containing
TB, 100 µg/mL ampilicillin, and 50 µM IPTG (to induce gene
expression) and allowed to grow 16 h at 30 °C. Glycerol was added to
the master plates (final concentration of 20%) and stored at -80 °C.
After 16 h of growth of the replicated plates, the plates were spun
down and the supernatant was removed. To the cell pellet a solution
of 1 mg/mL lysozyme, 0.1% Triton X-100, 20 mM each of the D-amino
acids to be screened, 1 mM NADP+, and 100 mM sodium carbonate,
pH 9.5 was added (20 µL/well).
The plates were allowed to shake at 200 rpm. During this time the
cells were lysed, and the enzyme was allowed to react with the substrate.
After 1 h the indicating dye, Nitro Blue Tetrazolium (0.15 mg/mL final
concentration), and the electron-transfer agent, phenazine methosulfate
(0.01 mg/mL final concentration) were added (20 µL/well). The plates
were monitored visually for wells changing from a pink to purple color.
Those wells that turned purple faster than the majority of the wells on
the plate (all wells eventually turned purple due to background
concentrations of NAD(P)H) were noted as being positives.20,21
D. Growth and Purification of DAPDH. The wild-type and mutant
DAPDH clones were grown and purified using the same procedure.
The DAPDH expressing E. coli were grown in 2.8 L baffled flasks
containing 1.2 L of TB and 100 µg/mL ampillicin. The flasks were
incubated at 30 °C and shaken at 180 rpm. After approximately 18 h
of growth, the cells were induced with 50 µM IPTG and continued to
be incubated at 30 °C and shaken at 180 rpm for an additional 18 h.
After this time the cells were harvested via centrifugation. The cells
Corynebacterium glutamicum bacteria were purchased from ATCC
(Manassas, VA). Platinum pfx DNA polymerase, pTrcHis2A expression
vector, and expression host Escherichia coli TOP10 cells were
purchased from Invitrogen (Carlsbad, CA). Taq DNA polymerase and
Taq DNA polymerase buffer were purchased from Roche (Indianapolis,
IN). Restriction endonucleases were obtained from New England
Biolabs (Beverly, MA). Oligonucleotides used for PCR amplification
were synthesized by IDT Inc. (Coralville, IA). Genomic DNA was
isolated using the MasterPure Complete DNA and RNA Purification
Kit. DNA ligations were performed using the Fast-Link DNA Ligation
Kit (EPICENTER, Madison, WI). Plasmid DNA was purified using a
PROMEGA (Madison, WI) Plasmid DNA Mini-prep Kit. Gene
sequencing was performed by the University of Florida, DNA Sequenc-
ing Lab (Gainesville, FL). Transformations were performed via
electroporation using the MicroPulser (Bio-Rad, Hercules, CA).
Methods. A. Cloning of the DAPDH Gene. The native DAPDH
gene (990 bp) was isolated from Corynebacterium glutamicum (ATCC
13032) using standard PCR amplification methods. Based on the pub-
lished sequence (NCBI accession No. Y00151) appropriate primers
(forward: 5′-AGTCCCCATGGGTACCAACATCCGCGTAGC-3′, re-
verse: 5′-AACTGCAGCCCTCGAGGGTTAGACGTCGCGTGCG-
ATCA-3′) were designed incorporating NcoI and XhoI as the 5′ and 3′
cloning sites, respectively. The forward primer also includes a glycine
(GGT) after the initial methionine to assist in cloning (all amino acid
numberings account for this inclusion). The PCR product was then
ligated to an expression vector (pTrcHis2A) cleaved with the same
(16) Raunio, R. P.; Straus, L. D.; Jenkins, W. T. J. Bacteriol. 1973, 115, 567-
573.
(17) Olsiewski, P. J.; Kaczorowski, G. J.; Walsh, C. J. Biol. Chem. 1980, 255
(10), 4487-4497.
(18) Misono, H.; Kato, I.; Packdibamrung, K.; Nagata, S.; Nagasaki, S. Appl.
EnViron. Microbiol. 1993, 59 (9), 2963-8.
(20) Mayer, K. M.; Arnold, F. H. J. Biomol. Screen 2002, 7 (2), 135-40.
(21) Williams, G. J.; Domann, S.; Nelson, A.; Berry, A. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100 (6), 3143-8.
(19) Billet, G. p-Hydroxyphenylpyruvic acid. Organic Syntheses; 1973; Col-
lective volume 5, p 627.
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10924 J. AM. CHEM. SOC. VOL. 128, NO. 33, 2006