The Crystal Structures of Apo and Complexed Saccharomyces cerevisiae GNA1 Shed Light on the Catalytic Mechanism of an Amino-sugar N-Acetyltransferase

Abstract

The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents. GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoAGlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 Å resolution, respectively. These structures not only reveal a stable, ␤-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level. Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.

Evolution of insect arylalkylamine N -acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti

“…Based on substrate specificities, a number of major GNAT superfamily proteins have been identified, including histone NAT (HAT), aminoglycoside NAT (AAC), aaNAT, and glucosamine 6-phosphate NAT (gpNAT). We chose representative structures from the foregoing enzymes for a structural comparison: a HAT from yeast (MYST; PDB code 1fy7) (38), a HAT from Tetrahymena (Gcn5/ PCAF; PDB code 1puA) (39), human HAT (p300/CBP; PDB code 3biy) (40), a different HAT from yeast (Rtt109; PDB code 3qm0) (41), an AAC6 from Enterococcus faecium (PDB code 1n71) (42), an AAC6 from Salmonella enterica (PDB code 1s3z) (43), an AAC2 from Mycobacterium tuberculosis (PDB code 1m4g) (44), a gpNAT from Trypanosoma (PDB code 3i3g) (45), human gpNAT (PDB code 3cxs) (46), a gpNAT from Aspergillus fumigatus (PDB code 2vxk) (47), a gpNAT from yeast (PDB code 1i21) (48), and SNAT from O. aries (PDB code 1cjw) (49). Superposition of five GNAT structures and three mosquito aaNATs revealed that the common fold cores are nearly universally conserved (Fig.…”

The Molecular Basis of the Expansive Substrate Specificity of the Antibiotic Resistance Enzyme Aminoglycoside Acetyltransferase-6′-Aminoglycoside Phosphotransferase-2“

“…In this case, an active site base may not be required considering that the pK a of its substrate (ϳ7.75) is sufficiently low that a significant proportion will be in the chemically competent form at physiological pH (33). A very complex picture of catalysis emerges from these findings where the molecular details of acetyl transfer in the GNAT family depend on the specific enzyme and/or substrate.…”

The Biology and Enzymology of ProteinN-Myristoylation

“…Removal of the C-terminal Met-454, Leu-455 of Nmt1p results in a 300 -400-fold reduction in the chemical transformation rate and converts the rate-limiting step of the enzyme from a step after chemical transformation to the transformation itself (47). In other GNAT members, a side chain carboxylate functions as a catalytic base to facilitate nucleophilic attack of a primary amino group on the thioester carbonyl of acetyl-CoA (50), although this role is assumed by the backbone carbonyl of Asn-134 in S. cerevisiae glucosamine-6-phosphate N-acetyltransferase 1 (51).…”