X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition

Eva Wolf, Jacqueline De Angelis, Ehab M. Khalil, Philip A. Cole, Stephen K. Burley

Research output: Contribution to journalArticle

Abstract

The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 Å resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA-S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site (cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyl-transferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.

Original languageEnglish (US)
Pages (from-to)215-224
Number of pages10
JournalJournal of Molecular Biology
Volume317
Issue number2
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Arylalkylamine N-Acetyltransferase
Acetyl Coenzyme A
Acetyltransferases
Catalysis
X-Rays
Catalytic Domain
Conserved Sequence
Enzymes
Transferases
Serotonin
Binding Sites
tryptamine
Inhibition (Psychology)

Keywords

  • Alternate conformations
  • Bisubstrate analog inhibitor
  • GCN5-related N-acetyltransferase
  • Melatonin biosynthesis
  • Serotonin N-acetyltransferase

ASJC Scopus subject areas

  • Virology

Cite this

X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition. / Wolf, Eva; De Angelis, Jacqueline; Khalil, Ehab M.; Cole, Philip A.; Burley, Stephen K.

In: Journal of Molecular Biology, Vol. 317, No. 2, 2002, p. 215-224.

Research output: Contribution to journalArticle

Wolf, Eva ; De Angelis, Jacqueline ; Khalil, Ehab M. ; Cole, Philip A. ; Burley, Stephen K. / X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition. In: Journal of Molecular Biology. 2002 ; Vol. 317, No. 2. pp. 215-224.
@article{2a45996898ad4189b9e4df6847e69d39,
title = "X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition",
abstract = "The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 {\AA} resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA-S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site (cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyl-transferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.",
keywords = "Alternate conformations, Bisubstrate analog inhibitor, GCN5-related N-acetyltransferase, Melatonin biosynthesis, Serotonin N-acetyltransferase",
author = "Eva Wolf and {De Angelis}, Jacqueline and Khalil, {Ehab M.} and Cole, {Philip A.} and Burley, {Stephen K.}",
year = "2002",
doi = "10.1006/jmbi.2001.5371",
language = "English (US)",
volume = "317",
pages = "215--224",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition

AU - Wolf, Eva

AU - De Angelis, Jacqueline

AU - Khalil, Ehab M.

AU - Cole, Philip A.

AU - Burley, Stephen K.

PY - 2002

Y1 - 2002

N2 - The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 Å resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA-S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site (cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyl-transferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.

AB - The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 Å resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA-S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site (cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyl-transferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.

KW - Alternate conformations

KW - Bisubstrate analog inhibitor

KW - GCN5-related N-acetyltransferase

KW - Melatonin biosynthesis

KW - Serotonin N-acetyltransferase

UR - http://www.scopus.com/inward/record.url?scp=0036299021&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036299021&partnerID=8YFLogxK

U2 - 10.1006/jmbi.2001.5371

DO - 10.1006/jmbi.2001.5371

M3 - Article

C2 - 11902838

AN - SCOPUS:0036299021

VL - 317

SP - 215

EP - 224

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -