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Insight into the 3D structure and substrate specificity of previously uncharacterized GNAT superfamily acetyltransferases from pathogenic bacteria.

ABSTRACT: Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening our knowledge of this diverse and important superfamily. In this work, we present the structural and kinetic analyses of two previously uncharacterized bacterial acetyltransferases - SACOL1063 from Staphylococcus aureus strain COL and CD1211 from Clostridium difficile strain 630. Our structures of SACOL1063 show substantial flexibility of a loop that is likely responsible for substrate recognition and binding compared to structures of other homologs. In the CoA complex structure, we found two CoA molecules bound in both the canonical AcCoA/CoA-binding site and the acceptor-substrate-binding site. Our work also provides initial clues regarding the substrate specificity of these two enzymes; however, their native function(s) remain unknown. We found both proteins act as N- rather than O-acetyltransferases and preferentially acetylate l-threonine. The combination of structural and kinetic analyses of these two previously uncharacterized GNATs provides fundamental knowledge and a framework on which future studies can be built to elucidate their native functions.

SUBMITTER: Majorek KA

PROVIDER: S-EPMC5127773 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Insight into the 3D structure and substrate specificity of previously uncharacterized GNAT superfamily acetyltransferases from pathogenic bacteria.

Majorek Karolina A KA Osinski Tomasz T Tran David T DT Revilla Alina A Anderson Wayne F WF Minor Wladek W Kuhn Misty L ML

Biochimica et biophysica acta. Proteins and proteomics 20161023 1

Members of the Gcn5-related N-acetyltransferase (GNAT) superfamily catalyze the acetylation of a wide range of small molecule and protein substrates. Due to their abundance in all kingdoms of life and diversity of their functions, they are implicated in many aspects of eukaryotic and prokaryotic physiology. Although numerous GNATs have been identified thus far, many remain structurally and functionally uncharacterized. The elucidation of their structures and functions is critical for broadening ...[more]

PMID: 27783928

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Project description:Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate=MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.

Dataset Information

Insight into the 3D structure and substrate specificity of previously uncharacterized GNAT superfamily acetyltransferases from pathogenic bacteria.

Publications

Insight into the 3D structure and substrate specificity of previously uncharacterized GNAT superfamily acetyltransferases from pathogenic bacteria.

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