Reconstitution and biochemical characterization of a new pyridoxal-5′-phosphate biosynthetic pathway

Article abstract of DOI:10.1021/ja042792t

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified. Copyright

Full text of DOI:10.1021/ja042792t

Published on Web 03/01/2005
Reconstitution and Biochemical Characterization of a New
Pyridoxal-5′-Phosphate Biosynthetic Pathway
Kristin E. Burns, Yun Xiang, Cynthia L. Kinsland, Fred W. McLafferty, and Tadhg P. Begley*
Department of Chemistry and Chemical Biology, Cornell UniVersity, Ithaca, New York 14853
Received November 30, 2004; E-mail: tpb2@cornell.edu
Pyridoxal-5′-phosphate (PLP, vitamin B6) is an essential cofactor
in all living systems.1 It plays an important role in amino acid and
carbohydrate metabolism and has recently been implicated in singlet
oxygen resistance.2 The biosynthesis of PLP in Escherichia coli
has been well studied. The enzymes PdxA and PdxJ catalyze the
conversion of 1-deoxy-D-xylulose-5-phosphate and 4-hydroxy-L-
threonine to pyridoxine phosphate, which is then oxidized to PLP
by PdxH.3 This pathway, however, is restricted to a relatively small
number of bacteria, and most bacteria, archaebacteria, fungi, and
plants do not contain PdxA/J/H homologues.4 In these organisms,
the highly conserved SNZ and SNO family of genes has been
implicated in PLP biosynthesis.4 Precursor labeling studies in yeast
suggest that carbons 2, 2′, 3, 4, and 4′ of PLP are derived from an
unidentified five carbon sugar, that carbons 5, 5′, and 6 are derived
from an unidentified triose, and that the pyridine nitrogen is derived
from the amide nitrogen of glutamine.5 In this communication, we
Figure 1. ESI-FTMS of freshly purified YaaD (charge state +32). (a) YaaD
as isolated. (b) Ribulose-5-phosphate treated YaaD.
identify the substrates for the SNZ and SNO family of proteins in
Bacillus subtilis (YaaD and YaaE; PLP synthase), describe the first
reconstitution of PLP biosynthesis by the major pathway, and
identify three new partial reactions catalyzed by the YaaD subunit
of PLP synthase.
The active site regions of the YaaE subunit of PLP synthase
shows high sequence similarity (41%) to glutamine amidotrans-
ferase, an enzyme that catalyzes the hydrolysis of glutamine to
glutamate and ammonia. This functional assignment has been
confirmed by the recent structure of YaaE6 as well as by the
reconstitution of its biochemical activity.7 A structural model for
the YaaD/YaaE complex has also been proposed in which the
ammonia generated at the YaaE active site is channeled to the active
site of YaaD where PLP formation occurs.6 The structural model
of YaaD shows a high level of similarity to imidazole glycerol-
phosphate synthase (HisF), the enzyme that catalyzes the formation
of the imidazole ring of histidine. One of the steps catalyzed by
this enzyme involves an amine addition to the C2 carbonyl of a
1-amino-ribulose-5-phosphate analogue (Scheme 1).8 This similarity
suggested that YaaD might also catalyze an amine addition to the
carbonyl group of ribulose phosphate. In addition, we have recently
found that 1-deoxy-D-xylulose-5-phosphate forms an imine with
thiazole synthase and that this imine is cleaved by an intramolecular
transimination later in the reaction sequence.9 Analogous imine
formation was a reasonable possibility for the YaaD catalyzed PLP
formation.
Figure 2. 1H NMR of the YaaD catalyzed isomerization of ribose-5-
phosphate to ribulose-5-phosphate 4. * indicates signals from ribulose-5-
phosphate. The triplet at 4.16 (H2 of 3) collapses to a doublet due to
deuterium exchange at C1.
(34 421.6 Da) that was 212 Da heavier than native YaaD (Figure
1a). The mass of this adduct is consistent with that expected for a
YaaD/pentulose phosphate imine. Treatment of the mixture with
ribose-5-phosphate 3 or with ribulose-5-phosphate 4 converted most
of the unmodified YaaD to the adduct as shown in Figure 1b. This
suggests that ribulose-5-phosphate is the bound carbohydrate. MS-
MS analysis of the YaaD adduct9 localized the imine to lysine 149,
which is absolutely conserved in the SNZ family of proteins. The
results were confirmed by demonstrating that the K149A mutant
did not form an adduct with ribulose-5-phosphate, suggesting that
the YaaD/ribulose-5-phosphate imine is an intermediate in the PLP
forming reaction.
Scheme 1
The observation that the YaaD adduct could be reconstituted with
equal ease from both ribulose-5-phosphate 4 and ribose-5-phosphate
3 suggested that YaaD catalyzed the interconversion of these two
compounds. This was confirmed by NMR analysis of a reaction
mixture containing ribose-5-phosphate and YaaD (Figure 2). Signals
with chemical shifts at 3.75 (m, 2H, H5), 3.88 (m, 1H, H4), and
4.29 (d, J ) 5.8 Hz, 1H, H3) confirm the formation of ribulose-
5-phosphate. It has been previously demonstrated that a ribose-5-
ESI-FTMS analysis of freshly isolated YaaD demonstrated the
presence of unmodified YaaD (34 209.0 Da) as well as an adduct
9
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10.1021/ja042792t CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
Figure 3. Rate of formation of PLP under various conditions. Ribose-5-
phosphate and glyceraldehyde-3-phosphate (A), ribulose-5-phosphate and
glyceraldehyde-3-phosphate (B), ribose-5-phosphate and dihydroxyacetone
phosphate (C), ribulose-5-phosphate and dihydroxyacetone phosphate (D).
phosphate isomerase mutant in yeast requires PLP for growth,10
suggesting that the yeast ortholog of YaaD (SNZ) may not have
this activity.
While previous labeling studies did not identify the triose
precursor to carbons 5, 5′, and 6 of PLP,5 dihydroxyacetone
phosphate or glyceraldehyde-3-phosphate 5 were the most likely
precursors and the stage was now set for attempting the in vitro
reconstitution of the biosynthesis. In the event, when ribulose-5-
phosphate, glyceraldehyde-3-phosphate, and glutamine were incu-
bated with PLP synthase, the reaction mixture turned yellow after
30 min at 37 °C and showed a UV-visible spectrum with an
absorbance maximum at 390 nm. HPLC analysis demonstrated the
formation of two reaction products that comigrated with PLP and
pyridoxal (PL) standards. A sample for NMR analysis was
generated by treating the reaction mixture with alkaline phosphatase,
followed by HPLC purification. Signals with chemical shifts of
2.35 (s, 3H, H2′), 4.86 (d, J ) 14.2 Hz, 1H, H5′), 5.10 (d, J )
14.2 Hz, 1H, H5′), 6.41 (s, 1H, H6), and 7.45 (s, 1H, H4′)
unambiguously identified the isolated product as pyridoxal.
The rate of formation of PLP, under various conditions, was
monitored by measuring the absorbance increase at 390 nm (Figure
3). Both ribulose-5-phosphate and ribose-5-phosphate, as well as
glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, are
substrates for PLP synthase. The absorption at 390 nm is dependent
on the YaaD concentration. The optimal pH for this reaction is
6-6.5. The initial lag of about 3 min cannot be explained currently;
however, it is important to note that it is observed under all
conditions tested so substrate isomerization does not play a role in
the lag.
The observation that dihydroxyacetone phosphate and glyceral-
dehyde-3-phosphate both were substrates suggested that PLP
synthase also has triose phosphate isomerase activity. To test this,
we incubated dihydroxyacetone phosphate with YaaD in D2O. NMR
analysis of this sample indicated that YaaD catalyzed complete
H-D exchange at C-1 of dihydroxyacetone phosphate, and no
glyceraldehyde-3-phosphate was detected. The absence of glycer-
aldehyde-3-phosphate from the reaction mixture is not unexpected
as the equilibrium constant lies in favor of dihydroxyacetone
phosphate.11 In addition, triose phosphate isomerase activity was
confirmed when we detected the formation of dihydroxyacetone
phosphate from YaaD and glyceraldehyde-3-phosphate by NMR.
We have demonstrated the first successful reconstitution of PLP
biosynthesis from glutamine, ribose-5-phosphate, and glyceralde-
hyde-3-phosphate. The identification of three partial reactions,
pentose isomerization, triose isomerization, and imine formation,
as well as the previous observation that dephospho-16 is a PLP
precursor12 allows us to propose a mechanism for PLP formation
as outlined in Scheme 2. In this proposal, imine formation between
ribulose-5-phosphate 4 and lysine 149 followed by enolization of
glyceraldehyde-3-phosphate gives 6. Loss of water followed by a
conjugate addition gives 8. Tautomerization to 9 followed by loss
of water and imine formation using ammonia, generated at the
glutaminase active site of YaaE, gives 11. Tautomerization followed
by a transimination gives 14. Substitution of the C4′ phosphate,
loss of water from C2′, and two tautomerization reactions complete
the biosynthesis.
Acknowledgment. We thank Olga Varsieva and Andrei Os-
terman for bringing this system to our attention. This research was
supported by grants from the NIH (DK44083 to T.P.B. and GM6609
to F.W.M.).
Supporting Information Available: Cloning and expression of
YaaD and YaaE, isomerization of ribose-5-phosphate, identification
of PL by NMR, rate of PLP formation, isomerization of dihydroxy-
acetone phosphate. This material is available free of charge via the
Internet at http://pubs.acs.org.
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