Characterisation of a hydroxymandelate oxidase involved in the biosynthesis of two unusual amino acids occurring in the vancomycin group of antibiotics.

Article abstract of DOI:10.1039/b103548g

ORF22 from the chloroeremomycin gene cluster has been cloned, expressed and characterised as a hydroxymandelate oxidase (HmO) that is involved in the formation of both (S)-4-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylglycine.

Full text of DOI:10.1039/b103548g

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Characterisation of a hydroxymandelate oxidase involved in the
biosynthesis of two unusual amino acids occurring in the vancomycin
group of antibiotics†
Tsung-Lin Li, Oliver W. Choroba, Elizabeth H. Charles, Alan M. Sandercock, Dudley H. Williams
and Jonathan B. Spencer*
Cambridge Centre for Molecular Recognition, Department of Chemistry, Lensfield Road, Cambridge,
UK CB2 1EW. E-mail: jbs20@cam.ac.uk
Received (in Cambridge, UK) 19th April 2001, Accepted 24th July 2001
First published as an Advance Article on the web 28th August 2001
ORF22 from the chloroeremomycin gene cluster has been
cloned, expressed and characterised as a hydroxymandelate
oxidase (HmO) that is involved in the formation of both (S)-
4-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylgly-
cine.
dioxygenase involved in the pathway to (S)-4-hydroxyphenyl-
glycine.3 This enzyme, 4-hydroxymandelic acid synthase
(HmaS), catalyses the production of 4-hydroxymandelate (4)
(4HMA) from 4-hydroxyphenylpyruvate (3) (4HPP) (Fig. 2).
Based on this discovery, it was postulated that ORF22, which
shows homology to a known glycolate oxidase4 (50% similarity
and 40% identity) might oxidise 4-hydroxymandelic acid to its
keto-acid derivative (5) followed by transamination, possibly
by ORF17, to give (S)-4-hydroxyphenylglycine (6) (Fig. 2).
During the preparation of this manuscript, Hubbard et al.
published a paper independently establishing parts of the results
presented here.5
The glycopeptide antibiotics vancomycin (1, Fig. 1) and
teicoplanin are currently the treatment of last resort against
methicillin-resistant Staphylococcus aureus.1 The continued
rise of vancomycin-resistant bacteria has heightened the need
for new therapeutic agents. Manipulation of the biosynthetic
gene cluster of glycopeptide antibiotics may be a potential route
to new antibiotics. To explore this approach we have set out to
elucidate the function of the genes involved in chloro-
eremomycin biosynthesis, another important member from the
vancomycin family (2, Fig. 1).2 In this communication we
report the overexpression of the protein coded for by orf22 from
the chloroeremomycin gene cluster, and its characterisation as a
flavin-dependent oxidase catalysing the conversion of 4-
hydroxymandelic acid and 3,5-dihydroxymandelic acid to their
corresponding keto acids.
(R)-4-Hydroxyphenylglycine, found at positions 4 and 5 of
the heptapeptide backbone of chloroeremomycin, is one of a
number of rare amino acids in glycopeptide antibiotics. The
peptide synthetase involved in the biosynthesis of chloro-
eremomycin contains epimerase domains for amino acids 2, 4
and 5, which suggests that (S)-4-hydroxyphenylglycine is the
precursor of (R)-4-hydroxyphenylglycyl units at positions 4 and
5. Recently we have determined that orf21 codes for a
In order to confirm the proposed pathway for the formation of
4HPG, orf22 was amplified by polymerase chain reaction
(PCR) and cloned into the expression vector pET28a(+)
(Novagen) as an N-terminal His6-tagged protein. The resulting
plasmid was used to transform the expression host E. coli
BL21(DE3). Overexpression was carried out at 37 °C in 1 L 2
3 YT medium with induction by isopropyl b- -thiogalactoside
D
(IPTG, 1 mM). The protein was recovered as an ammonium
sulfate precipitate and further purified on Ni2+-NTA agarose
resin. The resulting protein was then transferred into 50 mM
HEPES buffer, pH 7.6 using Millipore centrifugal filters. The
purified protein showed a single band on a SDS-PAGE gel. The
relative molecular mass was determined by ESI-MS to be 39863
5 Da which was in excellent agreement with that calculated
from the protein sequence (39864 Da). The enzyme contained a
cofactor which was released when the protein was denatured by
heat. This was determined to be FMN by HPLC analysis.
The enzyme (50 mg) was incubated with commercially
available racemic 4HMA (20 mM) in HEPES buffer (50 mM,
pH 7.6, 0.5 mL) in the presence of molecular oxygen, flavin
mononucleotide (FMN, 0.5 mM) and catalase (1600 IU) at
25 °C for 16 h. The reaction was then quenched with 100 mL 6
M HCl and extracted with ethyl acetate (3 3 1 mL). The
combined organic phases were dried with N2 gas and the
products converted to their methyl esters using diazomethane.
The mixture was analysed by ammonia chemical ionisation GC-
MS. The GC trace clearly shows that a new compound had been
produced with a retention time of 16.0 min (Fig. 3, the substrate
had a retention time of 16.0 min). The newly formed product
Fig. 1 Structures of vancomycin (1) and chloroeremomcyin (2).
† Electronic supplementary information (ESI) available: SDS-PAGE
analysis of HmO and molecular weight analysis of HmO protein by ESI-MS
and GC/CI-MS of the enzymatic conversion of 3,5DHMA with HmO. See
http://www.rsc.org/suppdata/cc/b1/b103548g/
Fig. 2 Proposed biosynthetic pathway for 4-hydroxyphenylglycine (6):
4HmaS, 4-hydroxymandelate synthase; 4HmO, 4-hydroxymandelate oxi-
dase; 4HpgT, 4-hydroxyphenylglyoxylate transaminase.
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Chem. Commun., 2001, 1752–1753
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b103548g

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Fig. 5 Visible spectra probing the generation of H2O2 during HmO
catalysis. a: enzymic reaction of horseradish peroxidase plus H2O2, o-
dianisidine and 4HMA, b: enzymic reaction of HmO plus o-dianisidine and
horseradish peroxidase, c: blank reaction omitting HmO.
Fig. 3 GC trace and CI mass spectrum of an enzyme reaction of 4HMA with
HmO after derivatisation by diazomethane showing 4-hydroxyphenyl-
glyoxylic acid (4HPGA, product) and 4-hydroxymandelic acid (4HMA,
substrate).
ular oxygen to oxidise the flavin after each reaction cycle with
the formation of hydrogen peroxide, whereas dehydrogenases
re-oxidised the flavin through electron transfer from proteins,
such as ubiquinine or cytochrome b5. To determine whether
HmO is an oxidase or dehydrogenase the incubation mixtures
were tested for the presence of hydrogen peroxide using a
coupled assay of horseradish peroxidase (HRP) and o-dianisi-
dine.11,12 HRP generates o-dianisidine radicals which combine
with 4HMA to give a product with an absorbance maximum at
550 nm (Fig. 5, trace a). When HmO was incubated with HRP
and o-dianisidine an increase in absorbance at 550 nm was
observed (Fig. 5, trace b). This determines that HmO is formally
a flavin dependent oxidase.
was identified as 4-methoxyphenylglyoxylate methyl ester
(identical retention time and fragmentation pattern to standard
material). This identifies orf22 as a hydroxymandelic acid
oxidase (HmO) and strongly suggests that it is involved in the
formation of (S)-4-hydroxyphenylglycine.
The stereochemistry of the substrate was elucidated using a
racemic mixture of 4HMA and determining the optical rotation
of the enantiomer remaining at the end of the incubation. Thus,
the 4-hydroxymandelic acid isolated from the reaction had a
25
negative optical rotation ([a]D 25.1°, c = 2.5, ethanol) which
25
corresponds to the same sign as synthetic (R)-4HMA ([a]D
210.2°, c = 0.98, H2O).6 The enzyme was also found to oxidise
commercially available (S)-mandelic acid but not (R)-mandelic
acid. These combined results lead to the conclusion that (S)-
4HMA is the natural substrate for the enzyme. Previous studies
had determined that (S)-3,5-dihydroxyphenylglycine, another
rare amino acid found in glycopeptide antibiotics, was formed
from acetate via a polyketide pathway.7,8 Analysis of the gene
cluster reveals orf27 to be homologous to a number of chalcone
synthases (polyketide synthases found mainly in plants). Recent
work suggests that the pathway to (S)-3,5-dihydroxyphenylgly-
cine involves the formation of 3,5-dihydroxyphenylacetic acid
by ORF279a followed by hydroxylation at the benzylic position,
oxidation to the keto-acid and transamination to (S)-3,5-dihy-
droxyphenylglycine. The last two steps in this pathway are the
same as those for (S)-4-hydroxyphenylglycine. Therefore it is
tempting to suggest that ORF22 might also participate in this
pathway and catalyse the oxidation 3,5-dihydroxyphenylman-
delate to its keto-acid derivative. To test this, racemic
3,5-dihydroxymandelic acid9b was incubated with HmO as
described previously (a shorter incubation time was used in this
case) and the products analysed by GC-MS. The results show
that 3,5-dihydroxymandelic acid is oxidised by the enzyme to
give 3,5-dihydroxyphenylglyoxylic acid. This provides strong
evidence that HmO is involved in the formation of both (S)-
4-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylglycine.
The reaction catalysed by HmO can therefore be summarised as
shown in Fig. 4.
In summary, we have identified the enzyme coded for by
orf22, as a hydroxymandelate oxidase (HmO) which carries out
the oxidation of both 4-hydroxymandelic acid and 3,5-hydroxy-
mandelic acid. This provides strong evidence that it is involved
in the pathway to both (S)-4-hydroxyphenylglycine and (S)-
3,5-dihydroxyphenylglycine and sheds new light on how these
important antibiotics are biosynthesised.
We thank the Royal Society for a Research Fellowship
(J. B. S.), the Ministry of Education, Taiwan, for a Research
Fellowship (T.-L. L.), the European Union for a TMR Marie-
Curie Research Fellowship (O. W. C), St. John’s College,
Cambridge for a Junior Research Fellowship (O. W. C.), and the
BBSRC for financial support. We thank Hwi Hong, in
particular, for her help in GC-MS analysis.
Notes and references
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3 O. W. Choroba, D. H. Williams and J. B. Spencer, J. Am. Chem. Soc.,
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4 P. Macheroux, V. Massey and D. J. Thiele, Biochemistry, 1991, 30,
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5 B. K. Hubbard, M. G. Thomas and C. T. Walsh, Chem. Biol., 2000, 7,
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7 S. J. Hammond, D. H. Williams and R. V. Nielsen, J. Chem. Soc., Chem.
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Flavin-dependent enzymes are normally divided into two
classes, oxidases and dehydrogenases.10 Oxidases use molec-
8 S. J. Hammond, M. P. Williamson, D. H. Williams, L. D. Boeck and
G. G. Marconi, J. Chem. Soc., Chem. Commun., 1982, 344.
9 (a) T.-L. Li, O. W. Choroba, H. Hong, D. H. Williams and J. B. Spencer,
Chem. Commun., accepted for publication; (b) A. M. Sandercock, E. H.
Charles, W. Scaife, P. N. Kirkpatrick, S. W. O’Brien, E. A.
Papageorgiou, J. B. Spencer and D. H. Williams, Chem. Commun.,
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10 R. B. Silverman, in The Organic Chemistry of Enzyme-Catalysed
Reactions, Academic Press, San Diego, 2000.
11 T. Betsche, D. Schaller and M. Melkonian, Plant Physiol., 1992, 98,
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12 I. G. Gazaryan, N. L. Klyachko, Y. K. Dulkis, I. V. Ouporov and A. V.
Levashov, Biochem. J., 1997, 328, 643.
Fig. 4 Oxidation of (S)-4-hydroxymandelate ((S)-4HMA) and (S)-3,5-di-
hydroxymandelate ((S)-4DHMA) to 4-hydroxyphenylglyoxylate by 4HmO
in the presence of FMN and molecular oxygen.
Chem. Commun., 2001, 1752–1753
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