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Applied and Environmental Microbiology, September 1999, p. 4028-4031, Vol. 65, No. 9
Tokyo Research Laboratories, Kyowa Hakko
Kogyo Co., Ltd., 3-6-6, Asahimachi, Machida, Tokyo 194-8533, Japan
Received 22 March 1999/Accepted 29 June 1999
Microbial proline 4-hydroxylases, which hydroxylate free
L-proline to
trans-4-hydroxy-L-proline, were
screened in order to establish an industrial system for
biotransformation of L-proline to
trans-4-hydroxy-L-proline. Enzyme
activities were detected in eight strains, including strains of
Dactylosporangium spp. and Amycolatopsis spp.
The Dactylosporangium sp. strain RH1 enzyme was partially
purified 3,300-fold and was estimated to be a monomer polypeptide with
an apparent molecular mass of 31 kDa by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on
the N-terminal amino acid sequence of the 31-kDa polypeptide were
synthesized in order to amplify the corresponding 71-bp DNA fragment. A
5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled
with digoxigenin as a probe for a genomic library of
Dactylosporangium sp. strain RH1 constructed in
Escherichia coli. One of the open reading frames found in
the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular
mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene.
The gene was expressed in E. coli as a fused protein with
the N-terminal 34 amino acids of the trans-4-Hydroxy-L-proline
is a chiral synthon that is useful for chemical synthesis of
pharmaceuticals (26). This compound is manufactured
industrially by acid hydrolysis of mammalian collagen, and no
biocatalytic method for production of hydroxyprolines has been described.
Hydroxyprolines have been found in certain proteins, such as collagen
(14), and in some peptide antibiotics, such as actinomycin (12) and etamycin (29). In mammalian systems,
L-proline is hydroxylated to
trans-4-hydroxy-L-proline by procollagen-proline dioxygenase (prolyl hydroxylase) (EC 1.14.11.2). This enzyme belongs to
the family of 2-oxoglutarate-dependent dioxygenases, which require
2-oxoglutarate and dioxygen as cosubstrates and ferrous ion as a
cofactor (2). It accepts only peptidyl proline as a
substrate, and free L-proline is not accepted. In contrast, proline 4-hydroxylase, which hydroxylates free L-proline to
free trans-4-hydroxy-L-proline, has been found
in a microbial system. However, the enzyme activity was found only
during biosynthesis of etamycin in Streptomyces
griseoviridus P-8648 (1, 15, 24). The proline
4-hydroxylase gene has never been cloned.
To determine the system which makes
trans-4-hydroxy-L-proline, we began by studying
the enzymatic conversion of L-proline, which is
industrially produced by fermentation, to
trans-4-hydroxy-L-proline through the action of
proline 4-hydroxylase. Since the previously reported activity was so
weak that the specific activity of the purified enzyme preparation was
907 pmol/min per mg of protein (15), extensive screening of
microbial proline 4-hydroxylases was necessary to obtain an enzyme with
high activity. Previously, we described a sensitive and efficient
screening system for the enzyme (20). Here, the results of
proline 4-hydroxylase screening, partial purification, cloning, and
expression of the proline 4-hydroxylase gene in Escherichia
coli are reported.
Chemicals.
Most chemicals were purchased from Nacalai Tesque
(Kyoto, Japan); L-proline was obtained from Kyowa Hakko
Kogyo Co., Ltd. (Tokyo, Japan).
Culture medium and cultural conditions.
HT medium and SR3
medium have been described previously (20). Df1 medium
consisted of 50 g of soluble starch per liter, 15 g of soy
bean meal per liter, 0.5 g of KH2PO4 per
liter, 0.5 g of MgSO4 · 7H2O per
liter, and 5 g of calcium carbonate per liter, and the pH was
adjusted to 7.0. All cultures were grown at 28°C. HT medium was used
to isolate microorganisms during the screening study. The
microorganisms were aerobically cultured with shaking for 2 days in 10 ml of SR3 medium. Then 1 ml of each culture was inoculated into 10 ml
of Df1 medium and cultured for 2 days with shaking. Cells were
collected by centrifugation and used for the whole-cell reaction study.
Df1 medium containing 1 g of L-Pro per liter was used
for a large-scale culture of Dactylosporangium sp. strain
RH1. RH1 cells were grown in a 30-liter fermentor containing 18 liters
of the medium for 3 days with agitation (385 rpm) and aeration (1 liter/liter/min). The cells were harvested by centrifugation and washed
once with a cold 0.85% (wt/vol) NaCl solution.
Taxonomic characterization of the strains.
The cultural
characteristics and morphology of strains RH1, RH2, RH3, RH4, and RH5
were determined by the methods of the International Streptomyces Project (27). The sugars in
whole-cell hydrolysates, menaquinones, phospholipids, and mycolic acids
were analyzed as described previously (4, 18, 19, 34).
Analytical methods.
Hydroxyprolines and proline were
detected by postcolumn derivatization with
7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride (NBD) (25),
as well as precolumn derivatization (16).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Microbial Proline 4-Hydroxylase Screening and
Gene Cloning
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-galactosidase 
-fragment.
The E. coli recombinant exhibited proline 4-hydroxylase
activity that was 13.6-fold higher than the activity in the original
strain, Dactylosporangium sp. strain RH1. No homology was
detected with other 2-oxoglutarate-dependent dioxygenases when
databases were searched; however, the histidine motif conserved in
2-oxoglutarate-dependent dioxygenases was found in the gene.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Enzyme assay. Cellular proline 4-hydroxylase activities were measured by the whole-cell reaction procedure. Whole-cell reactions were performed as described previously (20). Cell-free enzyme activities were assayed as follows. Each reaction mixture (250 µl) contained 80 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5), 4 mM L-proline, 8 mM 2-oxoglutarate, 2 mM ferrous sulfate, 4 mM L-ascorbic acid, and an enzyme preparation. The reaction mixtures were incubated at 35°C for 10 min, and then the reactions were terminated. The amount of trans-4-hydroxy-L-proline in each mixture was determined. For the assay in which recombinant E. coli was used, the reaction mixture contained 240 mM MES (pH 6.5), 12 mM L-proline, 24 mM 2-oxoglutarate, 8 mM ascorbic acid, 4 mM FeSO4, and cell extract. One unit of enzyme activity was defined as the amount of enzyme that hydroxylated 1 nmol of L-proline to trans-4-hydroxy-L-proline per 1 min.
Enzyme purification. Buffer A [50 mM N-tris-(hydroxymethyl)methyl-3-aminopropane sulfonic acid (TAPS) (pH 9.0) containing 2 mM dithiothreitol, 0.2 mM EDTA, and 20% (vol/vol) glycerol] and buffer B (50 mM TAPS [pH 8.0] containing 2 mM dithiothreitol, 0.1% [wt/vol] Tween 20, and 20% [vol/vol] glycerol) were used for enzyme purification. All procedures were performed at 0 to 5°C. RH1 cells suspended in buffer A were disrupted with a Dyno-mill (Willy A Bachofen Maschinenfabrik, Basel, Switzerland) and centrifuged (8,000 × g, 10 min). The supernatant was applied to a STREAMLINE DEAE column (Pharmacia), which was eluted with 0.2 M NaCl in buffer A. Then the pooled active fraction was applied to a DEAE-Sepharose column (Pharmacia), which was eluted with a linear 0 to 1 M NaCl gradient in buffer A. The pooled active fraction was applied to a Butyl Sepharose 4 Fast Flow column (Pharmacia) equilibrated with 3 M NaCl in buffer A. The column was washed stepwise with 3, 2, and 1 M NaCl in buffer A and then with buffer A. The pooled active fraction was applied to a HiTrap Phenyl Sepharose HP column (Pharmacia) equilibrated with 3 M NaCl in buffer A. The active fractions were eluted with 2 M NaCl in buffer A. Tween 20 was added to the active solution at a concentration of 0.1% (wt/vol). Then the solution was desalted with a PD-10 column (Pharmacia) and was applied to a Reactive Red 120-agarose column (type 3000-CL; Sigma Chemical Co., St. Louis, Mo.) equilibrated with buffer A containing 0.1% (wt/vol) Tween 20. Active fractions were eluted with a linear 0 to 1.5 M NaCl gradient in the same buffer. After it was desalted with a PD-10 column, the active solution was applied to a Reactive Blue 72-agarose column (Sigma) directly connected to a Resource Q column. The active fractions were eluted from the Resource Q column with a linear 0 to 0.2 M NaCl gradient in buffer B and used for characterization of the enzyme.
Cloning and nucleotide sequencing of the proline 4-hydroxylase
gene.
Degenerate oligoprimers F1
(5'-ATGCT[C/G]AC[C/G]CC[A/C/G/T]AC[A/C/G/T]GA)
and R1
(5'-GG[C/G]CC[C/G]AG[A/C/G/T]CC[A/G]TC[C/T]TC) corresponding to the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized and used for PCR with genomic DNA of
Dactylosporangium sp. strain RH1 prepared by the method of Hopwood et al. (10). The amplified fragments were cloned
into the SmaI site of pUC18. The cloned 71-bp fragment was
labeled with digoxigenin (DIG) by performing a PCR with a PCR DIG
labeling kit (Boehringer, Mannheim, Germany). Approximately 5.5-kb DNA fragments that hybridized to the DIG-labeled 71-bp fragment were recovered from XhoI-digested RH1 genomic DNA and ligated
with XhoI-digested 
ZAPII vector DNA (Stratagene). The
ligated DNA was packaged in vitro with Gigapack II Gold packaging
extract (Stratagene). The library was screened by performing
plaque hybridization with the DIG-labeled probe and a DIG detection kit
(Boehringer). Plasmid pRH71 was excised in vivo from a positive phage
by using E. coli SOLR (Stratagene) as the host.
Construction of expression plasmid for the proline 4-hydroxylase gene. Plasmid pRH71 was digested with SacI and blunted with a DNA blunting kit (Takara). A 2.4-kb fragment was recovered, digested with XbaI, and then ligated to EcoRV-XbaI-digested pBluescript II KS(+), which resulted in pES1-23a. E. coli DH1 was used as the host for expression of the proline 4-hydroxylase gene.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Screening for microbial proline 4-hydroxylases.
Screening for
proline 4-hydroxylases was done by the whole-cell reaction method as
described previously (20). The screening procedure was
focused mainly on actinomycetes grown on HT medium to which
hydroxyproline-containing actinomycin I or proline analogs were
sometimes added. More than 3,000 microbial strains isolated from soil
and etamycin-producing strains, including S. griseoviridus P-8648 (24), were examined. Five strains, RH1, RH2, RH3,
RH4, and RH5, were selected as proline 4-hydroxylase producers
(Table 1). Strains RH1, RH3, and RH4 were
identified as Dactylosporangium strains (7, 32)
based on the following taxonomic characteristics: branched substrate
mycelia on which claviform sporangia containing one row of two to four
spores were observed; meso-diaminopimelic acid was present
in the cell walls; whole-cell hydrolysates contained arabinose and
xylose as diagnostic sugars; and the major isoprenoid quinones were
MK-9(H6) and MK-9(H8). Strains RH2 and RH5 were identified as Amycolatopsis strains (7, 9). In
these strains, branched substrate and aerial mycelia were observed, but
spores, sporangia, synnemata, and other morphological characteristics were not; meso-diaminopimelic acid was present in the cell
walls; whole-cell hydrolysates contained arabinose and galactose; the major isoprenoid quinone was MK-9(H4);
phosphatidylethanolamine was present and no mycolic acids were present.
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Partial purification of proline 4-hydroxylase.
The proline
4-hydroxylase of Dactylosporangium sp. strain RH1 was
partially purified (Table 2). Although
the proline hydroxylase activity was enriched 3,300-fold, two major
bands, one at 80 kDa and one at 31 kDa, were identified in an SDS-PAGE
analysis of the purified enzyme preparation. The 31-kDa polypeptide
band on SDS-PAGE gels was thought to correspond to proline
4-hydroxylase based on the results of two different gel filtration
chromatography analyses performed with YMC-Pack Diol-2000
and TSKgel G-3000. The N-terminal amino acid sequence and two
internal amino acid sequences of the 31-kDa polypeptide were
determined to be MLTPTELKQY(R)EAGYLLIEDGLGP, XXNRTDNALPAQAAPRP, and AARDAT (where X is an amino acid that was not
identified and parentheses indicate an amino acid whose identity was
not certain).
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Cloning and expression of the proline 4-hydroxylase gene in E. coli. Plasmid pRH71, which contained the 5.5-kb XhoI fragment of RH1 genomic DNA, was isolated by the plaque hybridization method performed with the DIG-labeled 71-bp probe corresponding to the N-terminal amino acid sequence of the 31-kDa polypeptide. Sequencing of a 2.7-kb portion of the 5.5-kb fragment revealed that there were four open reading frames (ORFs) in the fragment, although ORF 3 was truncated and ORF 0 was not completely sequenced. ORF 1 is an 816-bp ORF that encodes a 272-amino-acid polypeptide (molecular mass, 29,715 daltons) containing amino acid sequences identical to the N-terminal and internal amino acid sequences of the 31-kDa polypeptide. The initiation codon, ATG, is preceded by a probable ribosome-binding site, AGGAG. The G+C content of ORF 1 is 74%, and 98% of the codons used in ORF 1 have G or C at the third position. A high G+C content and a codon usage bias are characteristics of genes from actinomycetes (33).
Expression plasmid pES1-23a, in which ORF 1 was expressed under the lac promoter as a fused protein consisting of the N-terminal 34 amino acids of the-galactosidase -fragment followed by 265 amino acids (amino acids 8 to 272) of ORF 1, was constructed (Fig. 1). Proline 4-hydroxylase activity was
detected in E. coli DH1 containing pES1-23a, and this
activity was 13.6-fold higher than the activity in
Dactylosporangium sp. strain RH1, indicating that ORF 1 encodes a proline 4-hydroxylase. The GenBank accession number of the
proline 4-hydroxylase structural gene is D78338.
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Comparison with other 2-oxoglutarate-dependent dioxygenases.
The search for sequences homologous to the deduced amino acid
sequences of ORF 0 to ORF 3 with the Genetics Computer Group package
program (Genetics Computer Group, Madison, Wis.) revealed no
significant similarities (i.e., the levels of identity were less than
40%) to the genes in the GenBank database, including the genes for
prolyl 4-hydroxylase and other 2-oxoglutarate-dependent dioxygenases.
We also detected no homology with proline 3-hydroxylase of
Streptomyces sp. strain TH1 (21). However, the
His-1 motif HXD (X indicates any amino acid), which is conserved
in the 2-oxoglutarate-dependent dioxygenase family (23),
seemed to be conserved in proline 4-hydroxylase at amino acids 109 to
111 (Table 3). The His-2 motif was not assigned to any part of the amino acid sequence of proline
4-hydroxylase (27, 31).
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DISCUSSION |
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Materials and Methods
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Discussion
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Hydroxyproline has been found in prokaryotic cells primarily in peptide antibiotics, such as actinomycin (12), etamycin (29), and telomycin (11); the only exception to this is a hydroxyproline-rich protein that has been found in Staphylococcus aureus (30). Proline 4-hydroxylase activity was found only during biosynthesis of etamycin in S. griseoviridus P-8648 (1, 15, 24). In the screening study reported here, five actinomycete strains were found to produce proline 4-hydroxylases, as were three etamycin-producing Streptomyces strains. These five actinomycete strains produced no etamycin as far as we could determine (Table 1). During the screening study we also found that Streptomyces and Bacillus strains produced proline 3-hydroxylases as well as telomycin-producing Streptomyces strains produce these enzymes (20). Our preliminary results suggested that the strains identified in this screening study produced peptidelike compounds containing hydroxyprolines other than etamycin and telomycin (20). Since hydroxyproline isomers, such as cis-4-hydroxy-D-proline, cis-4-hydroxy-L-proline, and trans-3-hydroxy-L-proline, are known to exist in nature along with trans-4-hydroxy-L-proline and cis-3-hydroxy-L-proline (14), there may be other proline hydroxylases with different regio- and stereospecificities.
Recently, Lawrence et al. described the purification and characterization of L-proline 4-hydroxylase from etamycin-producing S. griseoviridus P-8648 (15). The characteristics of proline 4-hydroxylase from S. griseoviridus P-8648 that were reported resembled those of the proline 4-hydroxylase described here with regard to the requirements for the reaction (i.e., Km values for L-proline and 2-oxoglutarate, which are substrates of the reaction and substrate specificity). However, the two enzymes were quite different in the following ways. First, Lawrence et al. reported that L-ascorbate inhibited 4-hydroxylation and proposed that L-ascorbate competed with 2-oxoglutarate for the same binding site on 4-hydroxylase. However, excess L-ascorbate never inhibited the 4-hydroxylase activity of Dactylosporangium sp. strain RH1. Second, the specific activity of partially purified 4-hydroxylase from Dactylosporangium sp. strain RH1 (2,080 nmol/min per mg of protein) was much higher than the reported activity of the purified 4-hydroxylase from S. griseoviridus P-8648 (907 pmol/min per mg of protein). This must be an advantage for industrial applications.
The proline 4-hydroxylase described here is classified as a 2-oxoglutarate-dependent dioxygenase because it requires 2-oxoglutarate and ferrous ion for a reaction. The deduced amino acid sequence of the enzyme has the same His motif that 2-oxoglutarate-dependent dioxygenase has (23). The HXD amino acid sequence in the His-1 motif is strongly conserved in the 2-oxoglutarate-dependent dioxygenase family, including proline 4-hydroxylase and proline 3-hydroxylase, as well as in isopenicillin N synthase, which is not a 2-oxoglutarate-dependent dioxygenase but requires ferrous ion and dioxygen. The histidine and aspartic acid residues in the His-1 motif and the histidine residue in the His-2 motif were found to take part in holding a ferrous ion in the enzyme by crystallographic studies of isopenicillin N synthase (27) and deacetoxycepharosporin C synthase (31). The histidine and aspartic acid residues are also conserved in both proline 4-hydroxylase and proline 3-hydroxylase (Table 3).
The proline 4-hydroxylase described here catalyzed highly regio- and stereospecific hydroxylation of L-proline. This rigidity of the reaction specificity makes the enzyme useful as a biocatalyst for the production of trans-4-hydroxy-L-proline from L-proline, which is industrially produced by fermentation. The enzyme also should be of some utility for preparing hydroxyproline isomers and some analogs. Cloning and expression of the proline 4-hydroxylase gene should lead to an effective method for producing trans-4-hydroxy-L-proline industrially.
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ACKNOWLEDGMENTS |
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We thank Ruriko Nishimura for capable technical assistance throughout the experiments. We also thank Keiichi Yano and Kazuhisa Uchida for N-terminal sequencing of the enzyme and technical advice.
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FOOTNOTES |
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* Corresponding author. Mailing address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahimachi, Machida, Tokyo 194-8533, Japan. Phone: 81-42-725-2555. Fax: 81-42-726-8330. E-mail: akio.ozaki{at}kyowa.co.jp.
Present address: Technical Research Laboratories, Kyowa Hakko Kogyo
Co., Ltd., 1-1, Kyowamachi, Hofu, Yamaguchi 747-8522, Japan.
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Materials and Methods
Results
Discussion
References
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