6
802 Biochemistry, Vol. 49, No. 31, 2010
Xiang et al.
subcluster 16 merges with subcluster 2. A sequence alignment of
subclusters 2 and 16 indicates that the conserved residues
responsible for substrate binding are retained, and thus the
enzymes contained within subcluster 16 are predicted to catalyze
the same chemical reaction as those found in subclusters 1 and 2.
Subcluster 4 contains the dipeptidases (such as Cc2672) that
recognize arginine and/or lysine at the C-terminus. At a BLAST
E-value of 10 , subcluster 15 merges with subcluster 4. How-
ever, this group of proteins does not share the canonical HxH
motif at the end of β-strand 1 but does exhibit an ExN motif.
This subcluster also lacks the HxH or HxY motif at the end of
β-strand 5. Finally, the conserved aspartate at the end of β-strand
6. Marti-Arbona, R., Fresquet, V., Thoden, J. B., Davis, M. L., Holden,
H. M., and Raushel, F. M. (2005) Mechanism of the reaction
catalyzed by iso-aspartyl dipeptidase from Escherichia coli. Biochem-
istry 44, 7115–7124.
7
8
9
. Xiang, D. F., Patskovsky, Y., Xu, C., Meyer, A. J., Sauder, J. M.,
Burley, S. K., Almo, S. C., and Raushel, F. M. (2009) Functional
identification of incorrectly annotated prolidases from the amidohy-
drolase superfamily enzymes. Biochemistry 48, 3730–3742.
. Xiang, D. F., Kumaran, D., Brown, A. C., Sauder, J. M., Burley,
S. K., Swaminathan, S., and Raushel, F. M. (2009) Functional
annotation of two new carboxypeptidases from the amidohydrolase
superfamily of enzymes. Biochemistry 48, 4567–4576.
. Cummings, J. A., Nguyen, T. T., Fedorov., A. A., Kolb, P., Xu, C.,
Fedorov, E. V., Shoichet, B. K., Barondeau, D. P., Almo, S. C., and
Raushel, F. M. (2010) Structure, mechanism, and substrate profile for
Sco3058: the closest bacterial homologue to human renal dipeptidase.
Biochemistry 49, 611–622.
-
70
7, required for the recognition of the guanidino group of arginine,
is replaced with a threonine. Therefore, we think that it is highly
unlikely that subcluster 15 catalyzes the hydrolysis of arginine-
containing dipeptides.
1
0. Cummings, J. A., Fedorov, A. A., Xu, C., Brown, S., Fedorov, E.,
Babbitt, P. C., Almo, S. C., and Raushel., F. M. (2009) Annotating
enzymes of uncertain function: the deacylation of
members of the amidohydrolase superfamily. Biochemistry 48, 6469–
481.
D-amino acids by
Subcluster 6 merges with subcluster 7 at an E-value cutoff of
6
-70
10
. The recognition elements for the hydrolysis of dipeptide
1
1. Hara, H., Masai, E., Katayama, Y., and Fukuda, M. (2000) The
4-oxalomesaconate hydratase gene, involved in the protocatechuate
substrates are retained in subcluster 6. The primary difference
between these two subclusters is the identity of the residue that
immediately follows β-strand 7, which appears to determine
C-terminal amino acid specificity for dipeptide substrates. In
subcluster 7 this residue is threonine, whereas this residue is
predominantly a glutamine in subcluster 6. The effect of this
substitution on the substrate specificity of subcluster 6 is un-
certain. Functional annotations of those subclusters (6, 11, 15,
and 16) most closely aligned with the experimentally annotated
subclusters (1, 2, 4, 5, and 7) are currently being addressed by a
combination of computational docking and compound library
screening.
4,5-cleavage pathway, is essential to vanillate and syringate degrada-
tion in Sphingomonas paucimolbillis SYK-6 hydration reaction.
J. Bacteriol. 182, 6950–6957.
2. Williams, L., Nguyen, T., Li, Y., Porter, T., and Raushel, F. M. (2006)
Uronate isomerase: a nonhydrolytic member of the amidohydrolase
superfamily with an ambivalent requirement for a divalent metal ion.
Biochemistry 45, 7453–7462.
3. Nguyen, T. T., Brown, T., Fedorov, A. A., Fedorov, E. V., Babbitt,
P. C., Almo, S. C., and Raushel, F. M. (2008) At the periphery of the
amidohydrolase superfamily: Bh0493 from Bacillus halodurans cata-
1
1
lyzes the isomerization of
chemistry 47, 1194–1206.
D-galacturonate to D-tagaturonate. Bio-
1
4. Nguyen, T. T., Fedorov, A. A., Williams, L., Fedorov, E. V., Li, Y.,
Xu, C., Almo, S. C., and Raushel, F. M. (2009) The mechanism of the
reaction catalyzed by uronate isomerase illustrates how an isomerase
may have evolved from a hydrolase within the amidohydrolase
superfamily. Biochemistry 48, 8879–8890.
ACKNOWLEDGMENT
1
1
5. Li, T., Iwaki, H., Fu, R., Hasegawa, Y., Zhang, H., and Liu, A. (2006)
R-Amino-β-carboxymuconic-ε-semialdehyde decarboxylase (ACMSD)
is a new member of the amidohydrolase superfamily. Biochemistry 45,
We thank members of the NYSGXRC protein production
team for providing purified samples of Sgx9260b and Sgx9260c.
6
628–6634.
6. Martynowski, D., Eyobo, Y. T., Yang, K., Liu, A., and Zhang, H.
2006) Crystal structure of alpha-amino-beta-carboxymuconate-
SUPPORTING INFORMATION AVAILABLE
(
Three figures showing F - F omit electron density maps for
epsilon-semialdehyde decarboxylase: insight into the active site and
catalytic mechanism of a novel decarboxylation reaction. Biochem-
istry 45, 10412–10421.
7. Liu, A, and Zhang, H. (2006) Transition metal-catalyzed monoxide
decarboxylation reactions. Biochemistry 45, 10407–10411.
8. Pegg, S. C.-H., Brown, S. D., Ojha, S., Seffernick, J., Meng, E. C.,
Morris, J. H., Chang, P. J., Huang, C. C., Ferrin, T. E., and Babbitt,
P. C. (2006) Leveraging enzyme structure-function relationships for
functional inference and experimental design: the structure-function
linkage database. Biochemistry 45, 2545–2555.
o
c
the active site of Sgx9260c complexed with N-methylphospho-
nate of
plexed with sulfate (Figure S2), and the active site of Cc2672
complexed with N-methylphosphonate of -arginine (Figure S3).
L-proline (Figure S1), the active site of Sgx9260b com-
1
1
L
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
9. Atkinson, H. J., Morris, J. H., Ferrin, T. E., and Babbitt, P. C. (2009)
Using sequence similarity networks for visualization of relationships
across diverse protein superfamilies. PLoS ONE 4, e4345.
0. Tyagi, R., Kumaran, D., Burley, S. K., and Swaminathan, S. (2007)
X-ray structure of imidazolone propionase from Agrobacterium
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