Identification of a lysine 4-hydroxylase from the glidobactin biosynthesis and evaluation of its biocatalytic potential

Article abstract of DOI:10.1039/c8ob02054j

We present the functional characterization of GlbB, a lysine 4-hydroxylase from the glidobactin biosynthetic gene cluster. Despite its narrow substrate specificity, GlbB is able to catalyze the hydroxylation of l-lysine with excellent total turnover number and complete regio- and diastereoselectivity. The synthetic utility of GlbB is illustrated by its use in the efficient preparation of a key dipeptide fragment of glidobactin.

Full text of DOI:10.1039/c8ob02054j

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Identification of a lysine 4-hydroxylase from the
glidobactin biosynthesis and evaluation of its
biocatalytic potential†
Cite this: DOI: 10.1039/c8ob02054j
Received 21st August 2018,
Accepted 8th October 2018
Alexander Amatuni
and Hans Renata
*
DOI: 10.1039/c8ob02054j
rsc.li/obc
We present the functional characterization of GlbB,
a lysine Pirrung, and others have provided initial validation towards
4-hydroxylase from the glidobactin biosynthetic gene cluster. the use of this natural product family as antineoplastic
Despite its narrow substrate specificity, GlbB is able to catalyze the agents.4,7
hydroxylation of L-lysine with excellent total turnover number and
Biogenetically, the macrolactam core of 1 and 2 arises from
complete regio- and diastereoselectivity. The synthetic utility of sequential condensations of 4-hydroxylysine, L-alanine, and
GlbB is illustrated by its use in the efficient preparation of a key malonyl-CoA catalyzed by modules of nonribosomal peptide
dipeptide fragment of glidobactin.
synthetases. In 2007,8 Dudler and co-workers identified the
biosynthetic gene cluster responsible for the production of gli-
dobactin from a soil bacterium belonging to Burkholderiales.
Eight genes, named glbA–H, were found to be crucial for the
The syrbactins are a family of hybrid peptide-polyketide
natural products that share
a 12-membered unsaturated
macrolactam core (Fig. 1A).1 Originally isolated as virulence
factors in plant-pathogen interactions, the syrbactins were
found to boost virulence by targeting the eukaryotic 20S pro-
teasome core particle.2 With reported IC50 values of <20 nM,
two members of this family, glidobactin A (1) and cepafungin I
(2) represent some of the most potent natural proteasome
inhibitors isolated to date.3 Crystallographic evidence suggests
that inhibition occurs primarily by covalent binding of the
α,β-unsaturated lactam to the chymotrypsin-like threonine
residue in the β5-subunit,2–4 akin to the mode of action of
several other proteasome inhibitors such as epoxomicin and
salinosporamide A.5 Proteasome inhibitors have recently
attracted attention as potential antitumor agents due to their
ability to disrupt the regulated degradation of pro-apoptotic
factors in malignant cells.6 At present, three proteasome
inhibitors, namely bortezomib, carfilzomib, and ixazomib,
have been approved by the FDA for the treatment of multiple
myeloma, and a few others are undergoing clinical trials.
Despite these developments, new proteasome inhibitors still
need to be advanced due to the potential emergence of drug
resistance and the syrbactins hold significant promise as such
candidates. Towards this end, total syntheses efforts and sub-
sequent medicinal chemistry development by Ichikawa,
Fig. 1 (A) Structures of glidobactin A (1) and cepafungin I (2), members
of the syrbactin family of natural products with potent inhibitory activity
against the 20S proteasome core. (B) Biosynthetic gene gluster of glido-
bactin in Polyangium brachysporum and predicted functions of the
respective genes in the formation of 1.
The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458, USA.
E-mail: hrenata@scripps.edu
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02054j
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biosynthesis of glidobactin (Fig. 1B). Two of the genes, glbC
and glbF, were assigned to encode for the NRPS modules
responsible for the formation of the macrolactam core struc-
ture. Of the remaining six, glbA, glbD, and glbE are thought to
encode for proteins involved in the export and regulation of
the metabolite production. GlbH shares homology with FAD-
dependent nitrodioxygenases and GlbB contains a conserved
domain within the DUF2257 family, hitherto a family of
uncharacterized conserved bacterial proteins. Though it is
likely that either of the latter two genes is responsible for the
production of 4-(OH)-Lys, the exact biosynthetic origin of this
motif was never elucidated on a molecular level.
In surveying the literature, several lines of evidence seem to
point to the role of GlbB in the production of 4-(OH)-Lys.
Firstly, deletion of glbH from the producer strain by Dudler led
to only reduction and not total abolition of glidobactin pro-
duction.8 A glbB disruption mutant, however, was found to be
completely incapable of producing glidobactin. A homologous
syrbactin synthetase from Photorhabdus luminescens, despite
containing only homologs of glbB–G, is capable of producing
glidobactin A when expressed in Pseudomonas putida.9 Finally,
while the function of DUF2257 (now known as PF10014) was
unknown at the time of Dudler’s work, subsequent investi-
gation by Ogawa and co-workers revealed that it belongs to the
iron- and α-ketoglutarate-dependent (Fe/αKG) superfamily of
enzymes and that its members are capable of hydroxylating
free-standing aliphatic amino acids.10 Indeed, a homology
model of GlbB, built using a solved crystal structure of a
member of PF10014 from M. petroleiphilum as a template,11
revealed the conserved His1-X-Asp-Xn-His2 metal binding triad
consisting of His188, Asp190, and His251. On the opposite
side of the pocket, an arginine residue (Arg267) is likely
involved in the binding of αKG via salt bridge formation. The
homology model also suggests a hydrophilic pocket formed by
Asp73, Arg82, Gln115, and Arg128, which likely forms key
polar contacts with the amino acid substrate (Fig. S1†). Based
on these observations, we postulate that GlbB might be
responsible for the C4 hydroxylation of L-lysine in the biosyn-
thesis of glidobactin. Recent results from our laboratory have
argued for the general utility of Fe/αKG enzymes as enabling
tools in the production of valuable building blocks for the syn-
thesis of complex bioactive molecules.12 Hypothesizing that
selective C4 hydroxylation of L-Lys can be leveraged for the che-
moenzymatic synthesis of syrbactins and related analogues, we
set out to functionally characterize GlbB.
Fig. 2 LCMS profile of L-Lys hydroxylation (a) in the presence of GlbB,
FeSO4, αKG, and ascorbic acid; (b) in the absence of GlbB; (c) in the
absence of FeSO4; (d) in the absence of αKG.
reaction product as 4, thus establishing the role of GlbB as
lysine 4-hydroxylase in the biosynthesis of glidobactin.
To investigate the utility of GlbB as a hydroxylation biocata-
lyst, we tested its activity against a suite of free amino acid sub-
strates. Among the 20 substrates tested (Fig. 3 and Fig. S4†),
only L-Lys, L-Leu and L-Met yielded the corresponding hydroxy-
lation products. Hydroxylation of L-Lys proceeded with com-
plete diastereoselectivity and high total turnover number
(5900 TTN). Similar to several other members of the PF10014
family, GlbB can catalyze the C4 hydroxylation of L-Leu to 5,
Codon optimization and over-expression of GlbB as an
N-His-tagged protein was found to provide good yield of
soluble enzyme (Fig. S3†). In vitro assays with recombinant
GlbB were next performed to test its ability to hydroxylate L-Lys
(Fig. 2). In the presence of FeSO4, α-ketoglutarate (αKG), and
Fig. 3 Substrate scope of amino acid hydroxylation with GlbB.
Conditions for total turnover number (TTN) measurement: free amino
acid (20 mM, 1 equiv.), αKG (50 mM, 2.5 equiv.), L-ascorbic acid (10 mM,
ascorbic acid, reaction of L-Lys with GlbB was observed to 0.5 equiv.), FeSO4 (1 mM, 0.05 equiv.), GlbB (0.01–0.15 mol% enzyme
loading), kPi buffer (pH = 8.0, 50 mM, 3 mL total volume), 12 h at 20 °C.
Yields and TTNs were determined by NMR analysis calibrating against an
internal standard of known concentration. Color legend for substrate
generate a new product with m/z = 163, corresponding to one
hydroxylation event. Omission of either FeSO4 or αKG from the
reaction led to abolition of catalytic activity, confirming the
table: green – excellent substrate, orange – moderate substrate, grey –
nonsubstrate. ^Reaction was carried out with 0.01 mol% GlbB. #Reaction
dependency of the enzyme on these components for activity.
Subsequent NMR characterization verified the identity of the was carried out with 0.15 mol% GlbB.
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albeit with only moderate TTN. Productive oxidation could
also be observed with L-Met to provide the corresponding sulf-
oxide product 6 with 330 TTN and ca. 10 : 1 dr. Additionally,
steady-state kinetic parameters were obtained for hydroxy-
lation of L-Lys, L-Leu, and L-Met (Fig. S5†). Consistent with the
observed TTNs, the catalytic efficiency of L-Lys hydroxylation of
L-Lys was found to be a few orders of magnitude higher than
that of L-Leu or L-Met, arising from significantly higher kcat
and much lower KM. With the exception of PolL—an Fe/αKG
involved in the biosynthesis of carbamoylpolyoxamic acid13—
previously characterized members of PF10014 have mostly
been limited to Fe/αKGs accepting only amino acid substrates
with aliphatic side-chains.10 To the best of our knowledge,
GlbB is the first member of this family capable of acting on
both polar and aliphatic amino acids. Prior to our work, the
two Fe/αKG lysine hydroxylases characterized thus far, KDO1
and KDO2,14 are classified as clavaminate-synthase-like Fe/
αKG dioxygenases (IPR014503 family), with minimal sequence
similarity to GlbB. In fact, despite catalyzing the hydroxylation
of the same carbon of L-Lys, KDO2 and GlbB share only 11%
sequence identity. The identification of lysine hydroxylation
activity within both PF10014 and IPR014503 families suggests
convergent evolution of this function from highly dissimilar
ancestral enzymes.
In agreement with GlbB’s unique substrate specificity, a
global survey of the PF10014 family, generated using the
sequence similarity network (SSN) approach, shows that GlbB
belongs to a distinct cluster separate from previously character-
ized members of PF10014 (Fig. 4). Notably, this cluster also
contains an Fe/αKG encoded by plu1881 (Uniprot accession
code: Q7N5R2, 48% sequence identity with GlbB) from the gli-
dobactin synthetase of P. luminescens.9 Assuming that enzymes
within this cluster exhibit similar substrate preference, our
analysis would predict that Plu1881 is also capable of catalyz-
Scheme 1 Application of L-Lys hydroxylation with GlbB in the synthesis
of a key dipeptide fragment (9) of glidobactin.
ing C4 hydroxylation of L-Lys. More importantly, the SSN of
PF10014 reveals the existence of several clusters with no func-
tionally characterized members. For example, we identify a
cluster containing HrmJ, an Fe/αKG that had been implicated
in the formation of 3-(trans-2′-nitrocyclopropyl)alanine from
L-Lys in the biosynthesis of hormaomycin.15 We anticipate that
functional characterization of enzymes belonging to these
lesser-known clusters will expand the breadth of chemical
reaction space spanned by the Fe/αKG dioxygenase
superfamily.
With an eye towards a chemoenzymatic total synthesis of
the syrbactins, we next assessed the utility of GlbB in prepara-
tive scale hydroxylation of L-Lys (Scheme 1). At 0.6 mmol scale,
full conversion of 3 to 4 could be obtained by using just
0.02 mol% of purified GlbB. Similar to other C4 and
C5 hydroxylated amino acids,12,14 4 was prone to undergo
intramolecular cyclization to gamma lactone
7 during
protection of the free amines. Further stirring of the crude
material from the amine protection in the presence of EDC
allowed the lactonization to proceed to full conversion, provid-
ing 7 in 60% isolated yield. Gratifyingly, 7 was found to
undergo facile amide bond formation with L-alaninol (8) at
room temperature to afford dipeptide 9 in 92% yield.16 This
sequence illustrates the synthetic utility of GlbB as a key
dipeptide fragment of glidobactin could be obtained in just
four simple steps.
Conclusions
Through a series of in vitro experiments, we have functionally
characterized a new Fe/αKG dioxygenase that is capable of cat-
alyzing selective C4 hydroxylation of L-Lys. These studies not
only shed light on the origin of the 4-(OH)-Lys motif in glido-
bactin, but also lay the groundwork for the development of a
chemoenzymatic strategy to access the syrbactin family of
natural products and related structural analogues.
Fig. 4 Sequence similarity network of PF10014 (100% identical Conflicts of interest
sequences are conflated, alignment score 60). Orange nodes represent
previously characterized Fe/αKG belonging to this family.
There are no conflicts to declare.
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Acknowledgements
Financial support for this work was generously provided by
The Scripps Research Institute and the National Institutes of
Health (Grant R35 GM128895). We thank the Shen lab, the
Kodadek lab, and the Roush lab for generous access to their
instrumentations.
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