Alanine racemase

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alanine racemase

The 1.45 a crystal structure of alanine racemase from Pseudomonas

aeruginosa, PDB 1rcq

Identifiers

EC number 5.1.1.1

CAS number 9024-06-0

Databases

IntEnz IntEnz view

BRENDA BRENDA entry

ExPASy NiceZyme view

KEGG KEGG entry

MetaCyc metabolic pathway

PRIAM profile

PDB structures RCSB PDB PDBe PDBsum

Gene Ontology AmiGO / EGO

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Ala_racemase_N

the 1.45 a crystal structure of alanine racemase from a pathogenic

bacterium, pseudomonas aeruginosa, contains both internal and

external aldimine forms

Identifiers

Symbol Ala_racemase_N

Pfam PF01168

Pfam clan CL0036

InterPro IPR001608

PROSITE PDOC00332

SCOP 1sft

SUPERFAMILY 1sft

[show]Available protein structures:

Ala_racemase_C

Identifiers

Symbol Ala_racemase_C
 Pfam PF00842

InterPro IPR011079

PROSITE PDOC00332

SCOP 1sft

SUPERFAMILY 1sft

[show]Available protein structures:

In enzymology, an alanine racemase (EC 5.1.1.1) is an enzyme that catalyzes the chemical
reaction
L-alanine D-alanine
Hence, this enzyme has one substrate, L-alanine, and one product, D-alanine.
This enzyme belongs to the family of isomerases, specifically
those racemases and epimerases acting on amino acids and derivatives. The systematic
name of this enzyme class is alanine racemase. This enzyme is also called L-alanine
racemase. This enzyme participates in alanine and aspartate metabolism and D-
alanine metabolism. It employs one cofactor, pyridoxal phosphate. At least two
compounds, 3-Fluoro-D-alanine and D-Cycloserine are known to inhibit this enzyme.
The D-alanine produced by alanine racemase is used for peptidoglycan biosynthesis.
Peptidoglycan is found in the cell walls of all bacteria, including many which are harmful to
humans. The enzyme is absent in higher eukaryotes but found everywhere in prokaryotes,
making alanine racemase a great target for antimicrobial drug development. [1] Alanine
racemase can be found in some invertebrates.Abe, H; Yoshikawa, N; Sarower, M. G.;
Okada, S (2005). "Physiological function and metabolism of free D-alanine in aquatic
animals". Biological & Pharmaceutical Bulletin. 28 (9): 1571
7. doi:10.1248/bpb.28.1571. PMID 16141518.
Bacteria can have one (alr gene) or two alanine racemase genes. Bacterial species with two
genes for alanine racemase have one that is continually expressed and one that is inducible,
which makes it difficult to target both genes for drug studies. However, knockout studies
have shown that without the alr gene being expressed, the bacteria would need an external
source of D-alanine in order to survive. Therefore, the alr gene is a feasible target for
antimicrobial drugs.[1]

Contents
[hide]

1Structural studies

2Proposed Mechanism

3References

4Further reading
Structural studies[edit]
To catalyze the interconversion of D and L alanine, Alanine racemase must position residues
capable of exchanging protons on either side of the alpha carbon of alanine. Structural
studies of enzyme-inhibitor complexes suggest that Tyrosine 25 and Lysine 39 are these
residues. The alpha-proton of the L-enantiomer is oriented toward Tyr265 and the alpha
proton of the D-enantiomer is oriented toward Lys39 (Figure 1).

Figure 1. Active site of Alanine Racemase. Tyrosine-265 and Lysine-39 are displayed with their
distances to the alpha-carbon of alanine, which is colored green and attached to PLP.

The distance between the enzyme residues and the enantiomers is 3.5A and 3.6A
respectively.[2] Structural studies of enzyme complexes with a synthetic L-alanine analog, a
tight binding inhibitor [3] and propionate [4] further validate that Tyr265 and Lys39 are catalytic
bases for the reaction,.[3][5]
The PLP-L-Ala and PLP-D-Ala complexes are almost superimposability.[2] The regions that do
not overlap are the arms connected the pyridine ring of PLP and the alpha carbon of alanine.
An interaction between both the phosphate oxygen and pyridine nitrogen atoms to the
5phosphopyridoxyl region of PLP-Ala probably creates tight binding to the enzyme. [2]

Figure 2. Surface Diagram of Alanine Racemase. The two monomers are colored in blue and green.
The two reaction sites are colored in red.

The structure of alanine racemase from Bacillus stearothermophilus (Geobacillus

stearothermophilus) was determined by X-ray crystallography to a resolution of 1.9 A.[5] The
alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta
barrel at the N terminus, and a C-terminal domain essentially composed of beta-strand. A
model of the two domain structure is shown in Figure 2. The N-terminal domain is also found
in the PROSC (proline synthetase co-transcribed bacterial homolog) family of proteins, which
are not known to have alanine racemase activity. The pyridoxal 5'-
phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and
is covalently linked via an aldimine linkage to a lysine residue, which is at the C terminus of
the first beta-strand of the alpha/beta barrel.

Proposed Mechanism[edit]
Reaction mechanisms are difficult to fully prove by experiment. The traditional mechanism
attributed to an alanine racemase reaction is that of a two-base mechanism with a PLP-
stabilized carbanion intermediate. PLP is used as an electron sink stabilize the negative
charge resulting from the deprotonation of the alpha carbon. The two based mechanism
favors reaction specificity compared to a one base mechanism. The second catalytic residue
is pre-positioned to donate a proton quickly after a carbanionic intermediate is formed and
thus reduces the chance of alternative reactions occurring. There are two potential conflicts
with this traditional mechanism, as identified by Watanabe et al. First, Arg219 forms a
hydrogen bond with pyridine nitrogen of PLP.[5] The arginine group has a pKa of about 12.6
and is therefore unlikely to protonate the pyridine. Normally in PLP reactions an acidic amino
acid residue such as a carboxylic acid group, with a pKa of about 5, protonates the pyridine
ring.[6] The protonation of the pyridine nitrogen allows the nitrogen to accept additional
negative charge. Therefore, due to the Arg219, the PLP-stabilized carbanion intermediate is
less likely to form. Another problem identified was the need for another basic residue to
return Lys39 and Tyr265 back to their protonated and unprotonated forms for L-alanine and
vice versa for D-alanine. Watanabe et al. found no amino acid residues or water molecules,
other than the carboxylate group of PLP-Ala, to be close enough (within 4.5A) to protonate or
deprotonate Lys or Tyr. This is shown in Figure 3.[2]

Figure 3. Schematic diagram of the distance between Lys39, Tyr 265, and PLP-L-Ala in the active
site. All interactions shown are under 4.5 and therefore are capable of hydrogen bonding. Adapted
from Watanabe et al.

Based on the crystal structures of N-(5-phosphopyridoxyl) L- alanine (PKP-L-Ala ( and N-(5-

phosphopyridoxyl) D-alanine (PLP-D-Ala)
Watanabe et al. proposed an alternative mechanism in 2002, as seen in the figure 4. In this
mechanism the carboxylate oxygen atoms of PLP-Ala directly participates in catalysis by
mediating proton transfer between Lys39 and Tyr265. The crystallization structure identified
that the carboxylate oxygen of PLP-L-Ala to the OH of Tyr265 was only 3.6A and the
carboxylate oxygen of PLP-L-Ala to the nitrogen of Lys39 was only 3.5A. Therefore, both
were close enough to cause a reaction.

Figure 4: The mechanism is based on x-ray structures of PLP-L-Ala and PLP-D-Ala and molecular
orbital calculations done by Watanabe (4).

This mechanism is supported by mutations of Arg219. Mutations changing Arg219 to a

carboxylate result in a quinonoid intermediate being detected whereas none was detected
with arginine.[7] The arginine intermediate has much more free energy, is more unstable, than
the acidic residue mutants.[6] The destabilization of the intermediate promotes specificity of
the reaction,.[7][8]