Structural analysis of an open active site conformation of nonheme iron halogenase CytC3 - PubMed (original) (raw)

Structural analysis of an open active site conformation of nonheme iron halogenase CytC3

Cintyu Wong et al. J Am Chem Soc. 2009.

Abstract

CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.

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Figures

Figure 1

Figure 1

Production of 4-Cl-

l

-Aba by CytC1−3. CytC1 loads

l

-Aba-AMP onto the phosphopantetheine arm (wavy line) on CytC2. CytC3 chlorinates the tethered substrate to form 4-Cl-

l

-Aba. CytC1 is an adenylation domain (A) and CytC2 is a thiolation domain (T).

Figure 2

Figure 2

Overall structure of CytC3 dimer. (a) Crystallographic dimer is colored by molecule. The iron ligands: two protein residues (His118 and His240) and αKG are shown in stick representation, and waters in the active site are shown in a spherical representation. The ends of the disordered region are indicated as “missing loop”, which consists of residues 178−219. (b) Structural alignment of SyrB2 monomer (pink) and CytC3 dimer (blue). (c) Surface representation of CytC3 dimer showing access to active site on one face of the crystallographic dimer. Active site waters are colored in red; each monomer of CytC3 is colored yellow and blue. The ends of the disordered region are colored in green.

Figure 4

Figure 4

CytC3 active site region. (a) Open active site in CytC3 structure allows for molecules from crystallization solution to bind, shown in stereo view. A 2_F_o − _F_c composite omit map contoured at 1σ is shown in blue mesh around excess ligands (αKG and sulfate). Iron is shown in brown, and waters are shown in red. (b) An iron anomalous difference map contoured at 12σ is shown in brown mesh around the iron. The αKG is labeled with the numbering nomenclature for each oxygen atoms. (c) A 2_F_o − _F_c composite omit map contoured at 2σ is shown in blue mesh around the active site ligands.

Figure 5

Figure 5

Active site comparison for CytC3 and SyrB2. Iron is shown in brown, chloride ion is shown in green, and waters are shown in red. (a) Comparison between CytC3′s “open” active site (blue) and SyrB2’s “closed” active site (pink), where structures are aligned by F106, K108, H125, W150, T148, R253, and R259 of CytC3 with the corresponding residues in SyrB2. (b) Comparison of CytC3 (blue) and SyrB2 (pink) where structures are aligned by the two active site histidines. (c) Active site residues of SyrB2 and relevant distances. Dash lines indicate hydrogen bonding with labeled distances. (d) Active site residues of CytC3 and relevant distances. The corresponding hydrogen bond interactions between the protein and αKG as observed in SyrB2 are indicated by black dashes. New interactions observed between CytC3 and αKG are indicated by green dashes with labeled distances.

Figure 6

Figure 6

Chloride binding pocket of SyrB2 and CytC3. Iron is shown in brown, chloride ion is shown in green, and waters are shown in red. (a) Residues A118, F121, and the β-carbon of S231 form a nice hydrophobic pocket for chloride in the closed state of SyrB2 active site. (b) The corresponding residues, A120, F123, and S236, in the open state of the CytC3 active site are too far from the chloride binding site to form a hydrophobic pocket environment.

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