Degradation of pyrimidines in Saccharomyces kluyveri: Transamination of β-alanine

Article abstract of DOI:10.1080/15257770802145983

Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme β-alanine aminotransferase (SkPyd4p), highly homologous to eukaryotic γ-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase (SkUga1p) with 80% and SkPyd4p with 55% identity to S. cerevisiae GABA-AT. SkPyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for α-ketoglutarate (αKG), β-alanine (BAL) and γ-aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. SkUga1p accepts only αKG and GABA but not BAL, thus only SkPydy4p belongs to the uracil degradative pathway. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770802145983

Nucleosides, Nucleotides, and Nucleic Acids, 27:794–799, 2008
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802145983
DEGRADATION OF PYRIMIDINES IN SACCHAROMYCES KLUYVERI:
TRANSAMINATION OF β-ALANINE
K. D. Schnackerz,1 G. Andersen,1 D. Dobritzsch,2 and J. Pisˇkur1
1Cell- and Organism Biology, Lund University, Lund, Sweden
2Department. of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm,
Sweden
Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have iden-
2
tified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding en-
zyme β-alanine aminotransferase (SkPyd4p), highly homologous to eukaryotic γ -aminobutyrate
aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase
(SkUga1p) with 80% and SkPyd4p with 55% identity to S. cerevisiae GABA-AT. SkPyd4p is
a typical pyridoxal phosphate-dependent aminotransferase, specific for α-ketoglutarate (αKG), β-
alanine (BAL) and γ -aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving
two half-reactions and substrate inhibition. SkUga1p accepts only αKG and GABA but not BAL,
thus only SkPydy4p belongs to the uracil degradative pathway.
Keywords Aminotransferase; β-alanine; γ -aminobutyrate; gene duplication; Saccha-
romyces kluyveri
INTRODUCTION
In biological systems, β-alanine (BAL) plays a major role as a precur-
sor of pantothenic acid,[1] a constituent of dipeptides such as anserine and
carnosine in the brain[2] and as an intermediate in the catabolic pathway
of pyrimidines. β-Alanine is a structural analog of γ -aminobutyrate (GABA)
and glycine, two major inhibitory neurotransmitters.[3] Moreover, BAL itself
has been suggested to be involved in synaptic transmission. Recent stud-
ies revealed that BAL activates the same receptors on the membranes of
rat sacral dorsal commissural neurons as glyine.[4] In mammalian systems,
uracil is degraded to BAL in three consecutive enzymatic steps by dihy-
dropyrimidine dehydrogenase, dihydropyrimidine amidohydrolase and β-
alanine synthase. In contrast, microorganisms produce BAL mostly by direct
Address correspondence to Klaus D. Schnackerz, Peter-Haupt-Str. 72, 97080 Wuerzburg, Germany.
E-mail: schnacke@biozentrum.uni-wuerzburg.de
794

Pyrimidine Degradation in S. kluyveri
795
decarboxylation of L-aspartate.[5] Bacteria and plants can also degrade sper-
midine to BAL.[6]
Mammals degrade BAL to malonate semialdehyde (MSA) using either
BAL:α-ketoglutarate aminotransferase (BAL-AT) or D-3-aminoisobutyrate:
pyruvate aminotransferase (DAIB-AT). The produced MSA is subsequently
metabolized by semialdehyde dehydrogenase to acetyl-CoA.[7] In humans,
BAL-AT and GABA aminotransferase activity resides within a single
enzyme.[8]
Even though Saccharomyces cerevisae can synthesize BAL from polyamines,
it cannot use BAL as sole nitrogen source.[9] GABA, in contrast, is read-
ily used as sole nitrogen source by the action of GABA-AT and succinic
semialdehyde dehydrogenase (SSADH), encoded by UGA1 and UGA2,
respectively.[10] Several yeast species, like Saccharomyces kluyveri and Schizosac-
charomyces pombe can utilize BAL as sole nitrogen source.[11]
In this study, we present the identification and characterization of two S.
kluyveri enzymes involved in transamination of BAL and GABA, encoded by
the PYD4 and UGA1 genes.
MATERIALS AND METHODS
All chemicals and enzymes were purchased from Sigma (St. Louis, MO,
USA). Yeast nitrogen base without amino acids and ammonium sulfate were
obtained from Difco (BD Biosciences, San Jose, CA, USA) and chelating
Sepharose fast flow from Amersham Biosciences (Piscataway, NJ, USA). Oli-
gos were purchased from DNA Technology (Aarhus, Denmark).
Strains and Media
The yeast and bacterial strains used in this study were grown in YPD
medium (1% yeast extract, 2% bactopeptone, 2% glucose) or synthetic
defined medium (1% succinic acid, 0.6% sodium hydroxide, 2% glucose,
0.67% yeast nitrogen base without amino acids and ammonium sulfate) sup-
plemented with different nitrogen sources (0.5% ammonium sulfate, 0.1%
for all other types). For solid medium (plates) 2% agar was added. For plas-
mid rescue, E. coli strain XL1-Blue was used, and for cloning TOP10 and
for overexpression of fusion proteins, BL21 Star. All bacteria were grown at
37◦C in LB medium supplemented with ampicillin (100 mg × L−1).
Mutagenesis
S. kluyveri mutants were generated from strains Y156 and 159 with
ethyl methanesulfonate.[11] Mutagenized cells were plated and grown for
2–3 days at 25◦C. The plates were replicated onto new plates contain-
ing ammonium sulfate (control) or dihydrouracil (DHU) as sole nitrogen

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K. D. Schnackerz et al.
source. After 5–7 days at 25◦C, colonies were selected based on their in-
ability to grow on DHU plates. Putative mutants were tested for growth on
β-ureidopropionate (BUP), with BAL as sole nitrogen source.
Cloning, Expression, and Purification of SkPyd4p
The cloning, expression and purification are described in detail
elsewhere.[12]
Clones of succinic semialdehyde dehydrogenase (SSADH) and mal-
onate semialdehyde decarboxylase (MSADC) were kindly provided by M. D.
Toney (University of California at Davis) and C. P. Whitman (University of
Texas, Austin) and the proteins obtained as previously described.[13,14]
Spectrophotometric Measurements
All measurements were performed on a Varian Cary 3 UV/Visible spec-
trophotometer equipped with a temperature controlled automatic multi-
cell changer.
Enzyme Assays
In a continuous assay, the production of MSA by BAL–AT is coupled
with MSADC to produce acetaldehyde, which in a NADH-dependent alco-
hol dehydrogenase reaction generates NAD+ and ethanol. The MSADC-
generated acetaldehyde was monitored by observing the NADH deple-
tion at 340 nm.[12] In a continuous assay, succinic semialdehyde (SSA)
produced by Uga1p and Pyd4p was converted by the NADP+-dependent
SSADH to NADPH and succinate. The absorbance change at 340 nm was
monitored.[12]
Data Analysis
Kinetic data analysis was performed as described previously.[12]
RESULTS
Identification of the PYD4 Gene Encoding a β-Alanine
Aminotransferase
While screening mutagenized S. kluyveri cells grown on DHU, a mutant,
Y947, was isolated which could not use DHU, BUP (β-ureidopropionate) or
BAL as sole nitrogen source. This mutant behaved like a double mutant,
since both BUP and BAL can serve as a direct nitrogen source via the ac-
tion of β-alanine synthase and BAL-AT. However, complementation with the
genomic library yielded plasmid P733, which supported growth on all three

Pyrimidine Degradation in S. kluyveri
797
nitrogen sources (DHU, BUP, and BAL). An open reading frame, termed
PYD4 (DQ512721), encoding a protein with 55% identity at the protein
level to S. cerevisiae UGA1 gene product, GABA-AT, was identified. A BLAST
search of Sc UGA1 in the S. kluyveri genome revealed another homologous
gene, termed SkUGA1 (DQ512722). Its gene product has 80% identity to
Sc Uga1p, and 57% to SkPyd4p.
Sequence Analysis of the PYD4 Gene
Phylogenetic analysis of the homologous proteins from yeast shows that
PYD4 is only found in two other sequenced yeasts (Debaryomyces hansenii and
Candida albicans). Both yeasts also contain a putative UGA1 gene. A search
for β-alanine synthase (encoded by PYD3) and dihydropyrimidine amidohy-
drolase (encoded by PYD2) revealed that S. kluyveri is the only yeast carrying
all three PYD genes. C. albicans and D. hansenii lack PYD2, and Kluyveromyces
lactis has PYD2 and PYD3, but lacks PYD4. S. cerevisiae lacks PYD2, PYD3, and
PYD4.
Purification and Properies of SkPyd4p and SkUga1p
The purification of the His-tagged SkPyd4p and SkUga1p was accom-
plished after disruption of the respective cells in three steps using strep-
tomycin treatment, affinity chromatography on nickel-chelating Sepharose
and size exclusion gel filtration on Sepharose S-12. From 1 liter of cell cul-
ture about 20 mg of pure protein could be obtained. The molecular weight
of the subunits of both proteins was found to be about 56-57 kDa based on
SDS/PAGE gel. Purified SkPyd4p and SkUga1p had native molecular masses
of 111 (dimers) and 135/240 kDa (dimers/tetramers), respectively. The
spectra of purified SkPyd4p and SkUga1p show two maxima at 340 and 410
nm, indicating the equilibrium of the ketoenamine (410 nm) and enolim-
ine (340 nm) tautomers which can be shifted by changing the pH of the
protein solution.
Initial Velocity Studies
In the initial reaction of SkPyd4p, αKG and BAL (or GABA) are con-
verted to glutamate and MSA (or SSA), respectively. In a second reaction,
either the amount of MSA or SSA was determined. The substrate specificity
of SkPyd4p was tested and it could be shown that SkPyd4p accepts both BAL
and GABA as amino donors but with a preference for BAL. In contrast,
SkUga1p uses only GABA as substrate not BAL.
Both SkPyd4p and SkUga1p were subjected to a full kinetic analysis. Since
SkUga1p shows no activity towards BAL, only SkPyd4p was analyzed with
both substrates. When the initial velocity was determined at various αKG

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K. D. Schnackerz et al.
concentrations and several fixed levels of BAL, a pattern composed of par-
allel lines is seen at low reactant concentrations in a double reciprocal plot.
At high αKG concentrations, the linear lines are deflected upwards, indi-
cating substrate inhibition. Values of KM,BAL, KM,GABA and KM,αKG with 8.2
0.4, 1.8 0.2, 2.9 0.2 mM, respectively, were determined for SkPyd4p.
Since the maximal velocity for SkPyd4p (7.5 units/mg) is more than eight
times higher for BAL, SkPyd4p can be considered a BAL-AT rather than a
GABA-AT. SkUga1p exhibits KM,GABAand KM,αKG values of 3.2 0.2 and 0.22
0.01 mM, respectively. The obtained kinetic behaviour is consistent with
a ping-pong kinetic mechanism.
DISCUSSION
Mammals have only one gene encoding a GABA-AT which exhibits both
BAL-AT and GABA-AT activity. S. cerevisiae has a single gene encoding GABA-
AT (ScUGA1), while S. kluyveri has two Sc UGA1-like genes. Similarly, the
genomes of C. albicans and D. hansenii each contain an UGA1-like gene. This
study identifies that one of the two genes in S. kluyveri, PYD4, encodes a
BAL-AT involved in BAL degradation. Apparently, the original PYD4/UGA1
gene was duplicated in one yeast lineage. Later on, one of the duplicated
genes was lost in some descendant lineages, such as S. cerevisiae. The time
point for the gene duplication is estimated to be ∼200 million years ago,
when Yarrowia lipolytica split from the Saccharomyces/Candida/Debaryomyces
lineages.[15]
The pattern of an array of parallel lines for different fixed concentra-
tions of the second substrate is consistent with a ping-pong kinetic mech-
anism. All aminotransferases adhere to such a mechanism, where the keto
substrates exert substrate inhibition. Both SkPyd4p and GABA-AT from E. coli
show comparable substrate inhibition for αKG with inhibition constants of
28.3 6.7 and 10.2 2.3 mM,[14] respectively. Both SkPyd4p and SkUga1p
show a reaction mechanism involving two half-reactions.
Our study shows that PYD4/SkPyd4p is a part of the reductive pathway of
pyrimidine nucleic acid precursors (Figure 1).
FIGURE 1 Reductive degradation of pyrimidine nucleic acid percursors. Uracil is degraded via dihy-
drouracil, N-carbamyl-β-alanine, β-alanine to malonate semialdehyde by dihydropyrimidine dehydroge-
nase (1, encoded by PYD1), dihydropyrimidine amidohydrolase (2, encoded by PYD2), β-alanine syn-
thase (3, encoded by PYD3), and β-alanine aminotransferase (4, encoded by PYD4), respectively.[16]

Pyrimidine Degradation in S. kluyveri
799
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