Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate

Internal aldimine and stable l -aspartate external aldimine

Wait R. Griswold, Andrew J Fisher, Michael D. Toney

Research output: Contribution to journalArticle

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Abstract

The 1.8 Å resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and l-aspartate external aldimine forms are reported. The l- aspartate•deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with l-aspartate. This structure is compared to that of the α-methyl-l-aspartate•PLP external aldimine. Overlays with the corresponding pyridoxal 5′-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the l- aspartate•deazaPLP and α-methyl-l-aspartate•PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4′. In contrast, in the α-methyl-l-aspartate•PLP external aldimine, the ε-amino group of Lys258 is rotated ∼70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.

Original languageEnglish (US)
Pages (from-to)5918-5924
Number of pages7
JournalBiochemistry
Volume50
Issue number26
DOIs
StatePublished - Jul 5 2011

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Aspartate Aminotransferases
Aspartic Acid
Pyridoxal Phosphate
Crystal structure
Hydrogen
Hydrogen bonds
Anchors
Schiff Bases
Escherichia coli
Half-Life
1-deazapyridoxal 5'-phosphate
Catalytic Domain
Phosphates
Crystals
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate : Internal aldimine and stable l -aspartate external aldimine. / Griswold, Wait R.; Fisher, Andrew J; Toney, Michael D.

In: Biochemistry, Vol. 50, No. 26, 05.07.2011, p. 5918-5924.

Research output: Contribution to journalArticle

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title = "Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate: Internal aldimine and stable l -aspartate external aldimine",
abstract = "The 1.8 {\AA} resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and l-aspartate external aldimine forms are reported. The l- aspartate•deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with l-aspartate. This structure is compared to that of the α-methyl-l-aspartate•PLP external aldimine. Overlays with the corresponding pyridoxal 5′-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to {"}anchor{"} PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the l- aspartate•deazaPLP and α-methyl-l-aspartate•PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4′. In contrast, in the α-methyl-l-aspartate•PLP external aldimine, the ε-amino group of Lys258 is rotated ∼70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.",
author = "Griswold, {Wait R.} and Fisher, {Andrew J} and Toney, {Michael D.}",
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T1 - Crystal structures of aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate

T2 - Internal aldimine and stable l -aspartate external aldimine

AU - Griswold, Wait R.

AU - Fisher, Andrew J

AU - Toney, Michael D.

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N2 - The 1.8 Å resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and l-aspartate external aldimine forms are reported. The l- aspartate•deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with l-aspartate. This structure is compared to that of the α-methyl-l-aspartate•PLP external aldimine. Overlays with the corresponding pyridoxal 5′-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the l- aspartate•deazaPLP and α-methyl-l-aspartate•PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4′. In contrast, in the α-methyl-l-aspartate•PLP external aldimine, the ε-amino group of Lys258 is rotated ∼70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.

AB - The 1.8 Å resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and l-aspartate external aldimine forms are reported. The l- aspartate•deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with l-aspartate. This structure is compared to that of the α-methyl-l-aspartate•PLP external aldimine. Overlays with the corresponding pyridoxal 5′-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the l- aspartate•deazaPLP and α-methyl-l-aspartate•PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4′. In contrast, in the α-methyl-l-aspartate•PLP external aldimine, the ε-amino group of Lys258 is rotated ∼70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group.

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