Development of a novel L-histidine assay method using histamine dehydrogenase and a stable mutant of histidine decarboxylase

Article abstract of DOI:10.1016/j.ab.2018.12.017

L-Histidine analysis is essential in physiological research and clinical applications because L-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for L-histidine quantitation has not yet been established. Here, we describe a novel L-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify L-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).

Full text of DOI:10.1016/j.ab.2018.12.017

Accepted Manuscript
Development of a novel L-histidine assay method using histamine dehydrogenase
and a stable mutant of histidine decarboxylase
Hiroki Yamaguchi, Kunio Nakata, Moemi Tatsumi, Masayuki Sugiki, Hiroshi Miyano,
Toshimi Mizukoshi
PII:
S0003-2697(18)30997-7
https://doi.org/10.1016/j.ab.2018.12.017
YABIO 13214
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 5 October 2018
Revised Date: 19 December 2018
Accepted Date: 21 December 2018
Please cite this article as: H. Yamaguchi, K. Nakata, M. Tatsumi, M. Sugiki, H. Miyano, T. Mizukoshi,
Development of a novel L-histidine assay method using histamine dehydrogenase and a stable mutant
of histidine decarboxylase, Analytical Biochemistry (2019), doi: https://doi.org/10.1016/j.ab.2018.12.017.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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[Title page]
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Analytical Biochemistry
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Development of a novel
L-histidine assay method using histamine dehydrogenase and a
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stable mutant of histidine decarboxylase
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Hiroki Yamaguchia, Kunio Nakataa, Moemi Tatsumia, Masayuki Sugikia, Hiroshi
Miyanoa, Toshimi Mizukoshia,*
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a Institute for Innovation, Ajinomoto Co., Inc, Kawasaki, Kanagawa 210-8681, Japan
* Corresponding author at Institute for Innovation, Ajinomoto Co., Inc, Kawasaki,
Kanagawa 210-8681, Japan.
Fax: +81-44-210-5872
E-mail address: toshimi_mizukoshi@ajinomoto.com
Subject category: Enzymatic assays
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[Abstract]
-Histidine analysis is essential in physiological research and clinical applications
because -histidine concentrations in biofluids are associated with various diseases.
However, an enzymatic method for -histidine quantitation has not yet been established.
Here, we describe a novel -histidine quantitation assay using a combination of
histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes
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.
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Wild-type HDC was unstable and completely lost its activity within 50 days of storage
at 4°C in solution. We rationally designed a HDC C57S mutant with markedly
improved stability (storage at 4°C for over 200 days) without altering the enzyme’s
substrate specificity. Together with HDH, the HDC C57S mutant was applied to
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quantify -histidine concentrations in human plasma. The assay showed high precision
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(< 2.0% inter-assay variation) and high accuracy (< 5.8% deviation from the results of
LC/MS).
[Graphical abstract]
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[Highlights]
· A novel colorimetric assay for quantitating
proposed.
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-histidine using two types of enzymes is
· A rationally designed
days.
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-histidine decarboxylase C57S mutant was stable for over 200
· Using this assay,
and accuracy.
L
-histidine can be quantitated in human plasma with high precision
[Keywords]
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Enzymatic assay,
L
-histidine decarboxylase, histamine dehydrogenase, Photobacterium
phosphoreum, human plasma analysis
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[Abbreviations]
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HDC; L-histidine decarboxylase, HDH; histamine dehydrogenase, LC/MS; liquid
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chromatography-mass spectrometry, BCAA; branched-chain amino acids, BTR;
BCAA/tyrosine molar ratio, PLP; pyridoxal 5′-phosphate, PDB; Protein Data Bank,
1-methoxy PMS; 1-ethoxy-5-methylphenazinium methylsulfate, WST-8; water-soluble
tetrazolium salt-8
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1. Introduction
-Histidine is an essential amino acid and many studies have examined its
physiological significance. Associations between the -histidine concentration in blood,
mental fatigue, and various diseases have been reported in humans [1]. For example,
high levels of -histidine are associated with stress and psychological disorders, and low
-histidine levels are associated with epilepsy, Parkinson’s disease, and nerve deafness
[2–5]. Recently, it was reported that the -histidine concentration in human plasma
represents a predictive biomarker for relapse of ulcerative colitis in patients in
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remission [6]. Therefore, accurate quantitation of the
biofluid samples is an important goal.
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-histidine concentration in
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Many studies have attempted to analyze the
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-histidine concentration, typically
using high-performance liquid chromatography or capillary electrophoresis [7–9].
However, a barrier in some applications is that these methods require expensive
instruments, highly skilled operators, and lengthy analyses. By contrast, simple
enzymatic assays can rapidly detect the presence of biological compounds with high
substrate specificity. Several enzymatic amino acid assays have been established in
clinical fields. For example, -leucine dehydrogenase has been applied for measurement
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of the BTR (the branched chain amino acid (BCAA)/tyrosine molar ratio) and the Fisher
ratio (BCAA/aromatic amino acid molar ratio); BCAA concentrations in the blood of
patients with hepatic cirrhosis tend to decrease [10–13]. Moreover,
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-phenylalanine
dehydrogenase is broadly used as a screening test for phenylketonuria in infants [14–
16]. However, an enzymatic assay for quantitation of
selectivity has not yet been reported.
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-histidine with high substrate
To overcome this problem, we developed a colorimetric enzymatic method using
-histidine decarboxylase (HDC) and histamine dehydrogenase (HDH). In the initial
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reaction, -histidine is converted to histamine by HDC, and in the detection step,
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histamine is detected by colorimetric formazan dye formation in the presence of
1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS). We used HDH from
Rhizobium sp. 4-9 because of its high thermostability and substrate specificity [17]. To
develop a one-pot -histidine assay, we used HDC from Photobacterium phosphoreum
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because of its high activity in the pH range required for Rhizobium sp. 4-9 HDH
stability [18]. However, the poor stability of wild-type P. phosphoreum HDC was a
considerable problem for the assay, as it rapidly lost activity during storage [19]. Thus,
we designed HDC mutants with improved stability using site-directed mutagenesis. We
demonstrate that the -histidine concentration in human plasma can be easily and
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accurately quantitated using a combination of stability-engineered HDC and HDH.
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2. Materials and methods
2.1 Cloning of the HDC gene
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The amino acid sequence of P. phosphoreum HDC (Uniprot ID: Q1JU62) was reverse
translated to DNA using GENETYX software (GENETYX CORPORATION, Tokyo,
Japan). To enable cloning into the pET-16b vector (Merck, Darmstadt, Germany), the
sequences 5′-TATCGAAGGTCGTCAT-3′ and 5′-GGATCCGGCTGCTAACAA-3′ were
added to the 5′ and 3′ termini of the HDC sequence, respectively. This construct was
chemically synthesized (Thermo Fisher Scientific, Waltham, MA, USA) and the
full-length HDC gene was amplified by PCR using oligonucleotide primers 5′-
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TATCGAAGGTCGTCATATGACCACCC-3′
(sense)
and
5′-
TTTGTTAGCAGCCGGATCCTTAGGC-3′ (antisense). The pET-16b vector was digested
with NdeI and BamHI, and the HDC-encoding fragment was cloned into the linearized
vector using the Seamless Ligation Cloning Extract method [20–21]. To add a His6 tag
to the C-terminus of this construct, pET-16b/HDC was further PCR-amplified using the
oligonucleotide
primers
5′-GCCGCACTCGAGCACCACCACCACCACCACTGAGGATCCGGCTGCTAACAAAGC
CCGAAAGGAAGC-3′ (sense) and 5′-GGCTGCAATCATTTCACCG-3′ (antisense). The
fragment was ligated back into the pET16b vector using T4 DNA ligase (TOYOBO,
Osaka, Japan).
2.2 Heterologous expression and purification of HDC
Escherichia coli BL21 (DE3) cells were transformed with the pET16b/HDC/His6
construct, and isolated transformants were grown overnight in LB medium containing
100 µg/mL ampicillin at 37°C. Expression of the His6-tagged fusion protein was induced
using 1 mM isopropyl-β-D-thiogalactopyranoside at 16°C for 16 h. The cells were
washed in lysis buffer [50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 2 mM
dithiothreitol and 0.2 µM pyridoxal 5′-phosphate (PLP)] and sonicated with an
ultrasonic disintegrator (ISOLATOR 201M, Kubota, Tokyo, Japan). The extract was
centrifuged for 30 min at 15,000×g. The supernatant was applied to a Ni Sepharose 6
Fast Flow column (GE Healthcare, Buckinghamshire, England) and washed with 50
mM Tris-HCl, pH 8.0, containing 500 mM NaCl, 75 mM imidazole, and 0.2 µM PLP.
Bound proteins were eluted using 50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl,
500 mM imidazole and 0.2 µM PLP. The protein-containing solution was desalted by
10-fold dilution in 50 mM Tris-HCl, pH 8.0, applied to a RESOURCE Q column (GE
Healthcare) and eluted using a 0–1000 mM NaCl gradient. The fraction eluted at 250
mM NaCl was collected. Finally, the HDC protein was dialyzed against 50 mM Tris-HCl,
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pH 8.0, containing 500 mM NaCl and 20% (v/v) glycerol, and stored at either 4°C or
−80°C until use.
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2.3 Homology modeling
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To identify a suitable template for homology modeling, the primary sequence of HDC
(Uniprot ID: Q1JU62) was used to search the Protein Data Bank (PDB) using BLAST
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software [22]. The X-ray crystal structure of L-tyrosine decarboxylase at 2.1 Å
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resolution (PDB ID: 3F9T) was identified as the most suitable template. Homology
modeling was conducted using the MODELLER package in Discovery Studio 4.5 [23].
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2.4 Screening of HDC mutants
HDC mutants were created by site-directed mutagenesis using the Quick Change
Lightning kit (Agilent Technologies, Santa Clara, CA, USA), and E. coli BL21 (DE3)
cells were transformed with each mutant vector. Transformed cells were suspended in
lysis buffer (50 mM Tris-HCl, pH 8.0, containing 500 mM NaCl and 75 mM imidazole)
and sonicated using an ultrasonic disintegrator (BIOLUPTER, Cosmo Bio Co. Ltd,
Tokyo, Japan). Each HDC mutant protein was simply purified using a HisTrap HP
column (GE Healthcare) and eluted with 50 mM Tris-HCl, pH 8.0, containing 500 mM
NaCl and 500 mM imidazole. The eluted proteins were normalized to 0.2 mg/mL and
incubated at 4°C and 30°C for 15 min. The enzyme activity of each mutant was analyzed
by a colorimetric assay. The 50 µL reaction mixtures, containing 100 mM sodium
phosphate, pH 6.5, 300 µM L-histidine, and each HDC mutant protein were incubated
at 25°C for 1 h. Conversion of L-histidine to histamine was monitored at 37°C using a
histamine detection kit (Kikkoman Biochemifa, Tokyo, Japan) containing histamine
dehydrogenase, 1-methoxy PMS, and water-soluble tetrazolium salt (WST)-8, and
absorbance at 470 nm was measured using a microplate reader (Varioskan LUX,
Thermo Fisher Scientific). The concentrations of histamine dehydrogenase, 1-methoxy
PMS, and WST-8 were based on a recommended procedure.
2.5 Size exclusion chromatography
For the purpose of analyzing oligomeric state, the wild type and C18S mutant
purified according to the procedure in section 2.2 were applied to a Superdex 200
Increase 10/300 GL column (GE Healthcare) with 50 mM Tris-HCl, pH 8.0, containing
500 mM NaCl, respectively.
2.6 Evaluation of HDC activity
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To evaluate enzyme kinetic parameters, the 150 µL reaction mixture composed 100
mM sodium phosphate, pH 6.5, 2.0 × 10-3 units of HDC which was purified according to
the procedure in section 2.2, 1.3 × 10-3 units of HDH, 1-methoxy PMS, WST-8, and
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serially diluted
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-histidine. The concentrations of -histidine were 0, 16, 31, 63, 125, 250,
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500 µM and 1, 2, 4, 8, 16 mM. The concentrations of 1-methoxy PMS and WST-8 were
based on a recommended procedure of histamine detection kit. One unit of HDC was
defined as the amount of enzyme that produced 1 µmol of histamine per min at 37°C
and pH 6.5, and one unit of HDH was defined as the amount of enzyme that produced 1
µmol of 4-imidazolylacetoaldehyde per min at 37°C and pH 9.0 based on the histamine
detection kit. The molar absorption coefficient of formazan dye at 470 nm was 3.1 × 104
M-1· cm-1. Absorbance at 470 nm was measured every 20 seconds at 37°C using a
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microplate reader. To assess enzyme substrate selectivity,
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-alanine, glycine,
-phenylalanine, -cysteine,
-histidine, -methionine,
-tryptophan, each at a final
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-isoleucine,
-aspartic acid,
-asparagine, -glutamine,
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-glutamic acid,
-serine,
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-valine,
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-proline,
-lysine,
-threonine, and
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L-arginine,
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concentration of 100 µM, were added to the reaction mixture.
2.7 Evaluation of HDC stability
The stability of HDC which was purified according to the procedure in section 2.2
was measured by the same assay condition as that used for the screening of mutants.
Enzyme stability during storage was evaluated using an HDC solution stored in the
dark at 4°C. To evaluate HDC thermostability, enzyme (0.7 × 10-3 unit in 50 mM
Tris-HCl, pH 8.0, containing 500 mM NaCl) was incubated at 4, 20, 25, 30, 35, 40, 45, 50,
55, 60, or 70°C for 10 min. To evaluate HDC oxidative stability, enzyme [0.7 × 10-3 unit
in 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, containing 0.001% (w/v) hydrogen peroxide
or 100 µM ascorbic acid] was incubated at 30°C for 15 min. For pH dependence, the
HDC activities at pH 5.5–7.0 and pH 7.0–9.0 were measured in MES and Tris-HCl
buffer, respectively. The absorbance at 470 nm of reaction mixtures containing 100 µM
L
-histidine was measured using the same procedure described section 2.6.
2.8 Human plasma analysis
One pooled and two individual human plasma samples were analyzed. The pooled
plasma sample was prepared by mixing equal volumes of plasma samples from eight
male and seven female individuals (KOJIN BIO, Saitama, Japan). Individual plasma
samples from a single male (Lot No. BRH955206) and female (Lot No. BRH964462)
donor were also purchased from KOJIN BIO. Plasma samples were centrifuged to
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remove precipitates immediately prior to measurement. For the enzymatic assay, the
150 µL reaction mixture composed of 100 mM sodium phosphate, pH 6.5, 2.0 × 10-3 unit
HDC, 1.3 × 10-3 unit HDH, 1-methoxy PMS, WST-8, and the sample. The concentrations
of 1-methoxy PMS, and WST-8 were based on a recommended procedure of histamine
detection kit. To construct a standard curve, 0, 50, 100, or 150 µM L-histidine were
added to the reaction mixture with 10-fold dilution. The limit of detection (LOD) was
calculated as an average value plus three standard deviations of the reaction mixture with 0
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µM
L-histidine (n = 3). For determination of the
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-histidine concentration, plasma
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samples were added to the reaction mixture after 45-fold dilution. Absorbance at 470
nm was measured every 20 seconds at 37°C using a microplate reader. The absorbance
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at 420 seconds was used for calculation of the
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-histidine concentration in plasma
samples with the linear regression equation of the standard curve.
2.9 Determination of
spectrometry (LC/MS)
L
-histidine concentration by liquid chromatography-mass
For L-histidine quantitation by LC/MS, we used an UF-Amino Station
(SHIMADZU, Kyoto, Japan) with automated precolumn derivatization as described
previously [24].
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3. Results and Discussion
3.1 Enzyme selection for the L-histidine assay
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To quantitate the -histidine concentration, a combination assay using both HDC and
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HDH was developed (Fig. 1). HDH from Rhizobium sp. 4-9 has high thermostability and
substrate specificity [17]. The assay conditions were optimized for HDH, which is
known to be stable from pH 5.5–10.5 [17]. Studies have been conducted on HDC
enzymes derived from a number of bacterial species [25–29]. The activation mechanism
of HDCs from Gram-positive and -negative bacteria differs: HDCs from Gram-positive
bacteria have a pyruvoyl moiety in their active site, while those from Gram-negative
bacteria use PLP as a cofactor [30–32]. The pyruvoyl moiety of Gram-positive HDCs is
acquired by cleavage of the proenzyme into its mature form [33–35]. In the case of
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Lactobacillus saerimneri 30A, formation of the pyruvoyl group occurred slowly in vitro
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which could limit enzyme preparation [28]. By contrast, maturation of Gram-negative
HDCs is dispensable and thus we chose to use HDCs from Gram-negative species to
simplify assay development [30, 31]. To develop the one-pot assay using Rhizobium sp.
4-9 HDH, we surveyed HDCs from the standpoint of pH dependence. HDC from P.
phosphoreum, a Gram-negative marine bacterium, was reported to be active from pH
5.0–8.0 [18]. Thus, we focused our efforts on P. phosphoreum HDC because its pH range
overlapped that of Rhizobium sp. 4-9 HDH
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Fig. 1. Scheme for the colorimetric -histidine quantitation assay.
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3.2 Expression and purification of wild-type HDC
Previous work showed that P. phosphoreum HDC could be overexpressed in E. coli
with an N-terminal His6 tag [27]. In our study, HDC bearing His6 tags fused to both N-
and C-termini was produced because this construct showed two-fold improved yield
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compared with an N-terminally His6-tagged construct. The recombinant HDC protein (a
43-kDa monomer) was purified by Ni-NTA affinity and anion exchange column
chromatography after sonication (Fig. 2). The yield of recombinant HDC was 16.6 mg/L
as shown using the Bradford method.
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Fig. 2. SDS-PAGE analysis of recombinant HDC from P. phosphoreum.
Lane 1 is a protein marker (Nacalai Tesque, Kyoto, Japan); Lane 2 is purified HDC
expressed in E. coli BL21 (DE3) cells. The arrow indicates the position of the HDC band.
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3.3 Design of stable HDC mutants
For the development of practical enzymatic assay methods, the storage stability of
enzymes is an important factor [36]. P. phosphoreum HDC is unstable, and it was
previously reported that its activity was lost after storage for only 7 days at 4°C [19].
One of the aims of this work was to produce a more stable HDC mutant. We hoped to
achieve stability equivalent to or better than that of the commercial enzyme,
Aspergillus niger glucose oxidase, which maintained about 40% of its activity after
storage for 60 days at 4°C in solution [37].
To improve the storage stability of enzymes, attempts are often made to mutate
cysteine residues located at solvent-accessible areas of their three-dimensional
structures [38, 39]. However, the structure of P. phosphoreum HDC has not yet been
reported. Therefore, we constructed a homology model of HDC (Fig. 3). After conducting
a BLAST search, tyrosine decarboxylase (PDB ID: 3F9T) was identified as a suitable
template. Compared with P. phosphoreum HDC, this enzyme had 51.0% sequence
identity at positions 158–254 near the cofactor-binding site of the pyridoxal-dependent
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decarboxylase domain (residues 82–339) [40]; the overall sequence identity between the
two enzymes was 36.7%. Four cysteine residues (Cys18, Cys57, Cys319, and Cys340)
had higher side-chain solvent accessibility (>39%) than other residues in the homology
model of HDC (Table 1). Previous reports demonstrated that substitution of cysteine
with serine could successfully improve enzyme storage stability [38, 41]. Thus, these
four cysteine residues were substituted with serine using site-directed mutagenesis.
The effects of Cys→Ser substitution on HDC storage stability were evaluated
using thermostability as a surrogate parameter [41], since evaluation of long-term
stability was not practical for the screening of mutants. We evaluated four cysteine
mutants (C18S, C57S, C319S, and C340S) by using the partially purified enzymes as
mentioned in section 2.4. Interestingly, the HDC C18S mutant completely lost its
enzymatic activity (Fig. 4); Cys18 was located within a solvent-accessible α-helix in the
N-terminal region of the homology model (Fig. 3). When we designed the cysteine
mutants, the solvent-accessible area was calculated using the monomer structure of the
homology model (Table 1). In the template structure of tyrosine decarboxylase (PDB ID:
3F9T), the enzyme was crystallized as a dimer, which was corroborated by gel shift
analysis (Fig. 5A) [42, 43]. HDC residues Cys18, Cys57, Cys319 and Cys340 correspond
to Asp25, Lys64, Lys330 and Cys351 of tyrosine decarboxylase, respectively. In the
structure of the tyrosine decarboxylase dimer, Asp25 was buried within surrounding
residues and mediated some inter- and intra-protomer interactions. As the result of size
exclusion chromatography, the molecular mass of the HDC C18S mutant was estimated
to be 820 kDa although that of wild-type was 70 kDa (Fig. S1). It was indicated that
HDC C18S mutant formed a weak aggregation which was inactive. Thus, we concluded
that the lack of activity of the HDC C18S mutant may have resulted from disruption of
active dimer formation.
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HDC C57S and C319S mutants showed residual activity (72.3% and 31.3%,
respectively) after incubation at 30°C for 15 min, while residual activity of wild-type
HDC was only 14.7% under the same conditions (right axis in Fig. 4). It is known that
residual activity after heat incubation is related with the storage stability [41].
Moreover, the relative activity of HDC C57S and C319S mutants was also dramatically
improved (left axis in Fig. 4). In particularly, HDC C57S mutant was 8.1 and 1.4 times
higher than that of wild-type and HDC C319S mutant, respectively. Relative activity
was evaluated by absorbance changes when HDH solution was added to the reaction
mixture after incubating for 60 min at 25°C because wild-type HDC maintained activity
for 60 min at 25°C (Fig. S2) The enzyme with high activity needs less volume and time
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to finish the reaction. HDC C57S mutant had the highest activity and residual activity
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so that would be suitable for the pragmatic assay. Therefore, we selected the HDC C57S
mutant for the following experiments.
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Fig. 3. Homology model of HDC.
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HDC and its nine encoded cysteine residues, shown as a ribbon (orange) and sphere
model, respectively. The homology model was built using the template structure (PDB
ID: 3F9T) in Discovery Studio 4.5. The active site (dotted line) was estimated from the
cofactor-binding site in the template structure.
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Table 1
Cysteine side chain solvent accessibility in the HDC homology model.
Position of Cys
Side chain
residue
18
solvent accessibility (%)
82.6
0
50
57
65.5
0.7
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282
319
330
340
0
15.3
94.1
8.7
39.7
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Side chain solvent accessibilities were calculated using Discovery Studio 4.5 (BIOVIA)
and presented as a percentage with respect to the Cys residue of an Ala-Cys-Ala
tripeptide.
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Fig. 4. Activity and residual activity of HDC cysteine mutants.
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The relative activity (black, left axis) and relative residual activity (gray, right axis)
after incubation at 30°C for 15 min of wild-type HDC are shown as solid and dotted
lines, respectively. The relative activity was calculated by comparison with the activity
of the wild type enzyme. The relative residual activity was calculated by comparison
with each activity without incubation at 30°C for 15 min. (n = 3, mean ± S.D.).
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(A)
(B)
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Fig. 5. Structure of tyrosine decarboxylase.
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(A) The dimeric structure of tyrosine decarboxylase (PDB ID: 3F9T) is shown as a
ribbon model (chain A in cyan and chain B in green). Asp25, Lys64, Lys330, and Cys351
are shown as a stick model (red). (B) The side chain solvent accessibilities of Asp25,
Lys64, Lys330, and Cys351 were calculated for the monomer (black) or dimer (gray)
structure using Discovery Studio 4.5.
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6
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3.4 Long-term storage stability of the HDC C57S mutant
After identifying the most thermostable HDC C57S mutant, we prepared the pure
enzyme by two step purification depending on procedure in section 2.2. At first, its
long-term stability was investigated during storage at 4°C in the dark (Fig. 6A). The
activity of wild-type HDC (control) dramatically decreased and the enzyme became
totally inactive within 50 days. By contrast, the HDC C57S mutant maintained its
activity over 200 days of storage. This improvement in long-term stability may be
sufficient to enable applications similar to those of related enzymes [44]. The
improvement in the long-term storage stability of the HDC C57S mutant was likely due
to two reasons. First, HDC C57S had improved thermostability at 30°C as mentioned
above (Fig. 4); moreover, the melting point of this mutant was 52.9°C compared with
40.7°C for the wild-type enzyme (Fig. 6B). Second, the substitution of Cys with Ser may
have improved the enzyme’s resistance to oxidation of thiol groups. Substitution of
cysteine residues has previously been shown to correlate with oxidative stability [45, 46].
In agreement with this hypothesis, the HDC C57S mutant was more resistant to
treatment with hydrogen peroxide than wild-type HDC (Fig. 6C). Wild-type HDC lost
half of its activity under oxidative conditions, while its activity was spared in the
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5
presence of antioxidant ascorbic acid. By contrast, the activity of HDC C57S mutant
was unaffected by the addition of hydrogen peroxide. These results showed that the
solvent-exposed Cys57 residue was extremely sensitive and that the HDC C57S mutant
was oxidation-tolerant.
(A)
(B)
6
7
(C)
(D)
8
9
Fig. 6. Stability of wild-type HDC and the HDC C57S mutant.
10
11
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14
15
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17
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19
(A) The stabilities of wild-type HDC and the HDC C57S mutant during storage at 4°C in
the dark were evaluated. The relative activity was calculated by comparison with the
activity on the first day. (n = 3, mean ± S.D.). (B) The thermostabilities of wild-type HDC
and the HDC C57S mutant were measured via their residual activities after heat
treatment for 15 min. The relative activity was calculated by comparison with the
activity at 4°C. (C) The oxidative resistance of wild-type HDC and the HDC C57S
mutant were measured. Prior to analysis, the enzymes were incubated with and
without hydrogen peroxide (H2O2) or ascorbic acid (AA). The relative activity was
calculated by comparison with the activity without H2O2 and AA. (n = 3, mean ± S.D.).
(D) The pH dependence of wild-type HDC and the HDC C57S mutant were measured at
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pH 5.5–9.0. The both MES and Tris-HCl buffer were used at pH 7.0 as mentioned in
section 2.7. The relative activity was calculated by comparison with the activity at pH
6.5 (n = 3, mean ± S.D.).
3
4
5
6
3.5 Substrate specificity of the HDC C57S mutant
7
Next, we assessed whether the C57S substitution affected the substrate selectivity of
HDC (Table 2). Both wild-type HDC and the HDC C57S mutant were able to act on
8
9
L
-histidine selectively among the 20 naturally-occurring amino acids. This specificity
was sufficient for quantitation of -histidine in crude biological samples.
The kinetic parameters of both wild-type HDC and the HDC C57S mutant are shown
in Table 3. The m and cat of HDC C57S mutant were half value than those of wild-type.
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11
12
13
14
15
16
17
18
19
20
21
L
K
k
From the model structural view, the Cys57 residue was found to be located at the
entrance of the substrate pocket in a homology model of the HDC dimer (Fig. S3). This
indicated that the smaller size of serine residue compared with cysteine residues, might
affect substrate access to the active site. To understand the detail of this mechanism,
one would need to study substrate recognition at the atomic level by determining the
structure of the
L
-histidine/HDC complex.
Table 2
Substrate selectivity of wild-type HDC and the HDC C57S mutant.
relative activity
relative activity
C57S
C57S
wild-type
wild-type
mutant
mutant
ND
His
Cys
Tyr
Asp
Asn
Glu
Pro
Ser
Gln
Arg
100%
1%
100%
1%
Thr
Met
Lys
Ala
Gly
Phe
Ile
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Leu
Val
Trp
ND
ND
ND
22
23
The enzyme activity when histidine was used as the substrate was set as 100%. ND, not
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4
determined. When histidine was used as substrate, the activities of wild-type HDC and
HDC C57S mutant were 0.09 U/mL and 0.10 U/mL, respectively.
Table 3 Kinetic parameters of wild-type HDC and HDC C57S mutant.
Km
kcat
(s-1)
2.81
1.46
kcat/Km
(mM)
(M-1·s-1)
wild-type
1.59 × 10-1
8.13 × 10-2
1.77 × 104
C57S mutant
1.79 × 104
5
6
To calculate
K
m and
k
cat values, the concentrations of
L
-histidine 0, 16, 31, 63, 125, 250,
500 µM and 1, 2, 4, 8, 16 mM.
7
8
3.6 Standard curve for
Using wild-type HDC and HDH in the assay condition shown in 2.8, we developed
the assay method for -histidine in human plasma whose concentration had been
L
-histidine quantitation
9
10
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13
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18
19
20
21
22
23
24
25
26
L
reported to be in the range of 63.8–97.9 µM [48] (Fig. 7). Also, in this study, we showed
that the HDC C57S mutant had higher storage stability than that of wild-type HDC
(Fig. 6A), and the substrate specificity and kcat/Km value of HDC C57S mutant was
similar to that of wild-type HDC (Table 2 and 3). As so, we evaluated whether the HDC
C57S mutant could be applicable of our method. As a result, the reaction time required
to consume 150 µM -histidine was 900 seconds for wild-type HDC and 420 seconds for
L
the C57S mutant, respectively (Fig. 7). In addition, the HDC C57S mutant had the high
activity over the pH range 6.0–8.5 (Fig. 6D). Because its pH range also overlapped that
of Rhizobium sp. 4-9 HDH, the one-pot
L
-histidine assay could be developed using HDC
-histidine, four concentrations of standard
C57S mutant and HDH. For quantitation of
L
solutions were added to reaction mixtures in the presence of both HDC C57S mutant
and HDH, and absorbance changes were monitored. Linear standard curves were
obtained with absorbance endpoints at 420 seconds in the range 0 to 150 µM
L
-histidine
(Fig. 8). The correlation coefficient was estimated to be 0.9982, and the limit of detection
was 1.9 µM. This standard curve covered the range of. -histidine concentrations in
L
human plasma.
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5
6
7
8
9
Fig. 7. Time course of the absorbance change with wild-type HDC and the HDC C57S
mutant.
Absorbance at 470 nm was monitored every 20 seconds for wild-type HDC (closed
circles) and the HDC C57S mutant (open squares). The each reaction mixture contained
2.0 × 10-3 unit wild-type HDC or the HDC C57S mutant. Wild-type HDC reached a
plateau at 900 seconds (dotted line) while the HDC C57S mutant did so at 420 seconds
(n = 3).
10
11
Fig. 8. L-histidine standard curve for the HDC C57S mutant.
12
13
14
15
16
17
18
The horizontal axis shows the -histidine concentration. The vertical axis shows the
L
absorbance change at 470 nm per 420 seconds determined by the enzymatic method
using the HDC C57S mutant (n = 3).
3.7 Analysis of human plasma
Three human plasma samples (A–C) were added to reaction mixtures to measure
L
-histidine concentrations without deproteination. These results were compared with
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those determined by LC/MS using an automated precolumn deviation method (Table 4)
[24]. The enzymatic assay was able to determine -histidine concentrations in human
L
3
plasma samples with high accuracy (≤ 5.8% deviation from the results of LC/MS) and
precision (≤ 2.0% inter-assay variation). Endogenous histamine did not interfere with
quantification because of its low concentration (reported as 1–4 nM) [49]. Thus, the
4
5
6
assay combining the HDC C57S mutant and HDH enables analysis of
physiological research and clinical applications.
L
-histidine for
7
8
9
Table 4 Quantitation of histidine in human plasma by LC/MS and a HDC C57S mutant
enzymatic assay.
10
plasma
A
B
C
1st
2nd
3rd
75.3
72.7
74.6
1.8
96.8
93.7
93.4
1.4
62.0
63.8
63.0
2.0
Histidine concentration
by HDC C57S mutant assay (µM)
CV (%)
Histidine concentration
by LC/MS (µM)
73.5
0.9
94.3
5.8
59.5
0.3
Rate of deviation (%)
11
12
Plasma A: pooled, B: individual male, C: individual female (n = 3)
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4. Conclusions
We have described a novel
L
-histidine quantification method based on a colorimetric
3
enzymatic assay using a mutant HDC with improved stability and HDH. The poor
stability of wild-type P. phosphoreum HDC and loss of activity during long-term storage
was improved by site-directed mutagenesis to introduce a C57S substitution. HDC
C57S mutant retained most of its enzymatic activity after more than 200 days of storage.
Improvements in enzyme thermostability and oxidative stability were inferred to
directly contribute to enhanced long-term storage stability. Using the HDC C57S
mutant in combination with HDC, the enzymatic assay described here was able to
4
5
6
7
8
9
10
11
12
quantitate
L
-histidine in human plasma with high accuracy and precision. This assay
could be useful in physiological research and clinical applications.
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5. Acknowledgments
We thank Makoto Otake, Shiori Kimura and Kazuki Noguchi for excellent technical
assistance. We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this
manuscript.
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