Structural and mechanistic studies of theorf12gene product from the clavulanic acid biosynthesis pathway

Abstract

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

In vivo functional analysis of a class A β-lactamase-related protein essential for clavulanic acid biosynthesis in Streptomyces clavuligerus

“…Of particular relevance to the current study is the product of orf12 (SCLAV_4187) from the CA-BGC of S . clavuligerus , which resembles class A β-lactamases and also contains similar S XXK, S DN and KAG amino acid motifs [25]. orf12 is co-transcribed with orf13 , which encodes a putative membrane transport protein (Fig 2A), and their relative arrangement also suggests possible translational coupling [23].…”

“…Studies have shown that orf12 is required for clavulanic acid, but not 5 S clavam production [23] and that the encoded protein lacks any detectible β-lactamase activity. Instead, heterologously expressed and purified Orf12 was shown to function as a cephalosporin esterase under in vitro conditions [25], due to which it is henceforth referred to as Cpe (for cephalosporin esterase).…”

“…The recently solved crystal structure of Cpe showed that in addition to a β-lactamase-like domain located in its C-terminus (residues 128–458), the protein also contains a previously unrecognized N-terminal domain (residues 1–127) resembling those found in steroid isomerases and polyketide cyclases [25]. In addition, two molecules of clavulanic acid were found to be bound non-covalently to Cpe when crystals of the protein were soaked in a solution of the metabolite during structural studies [25].…”

“…The recently solved crystal structure of Cpe showed that in addition to a β-lactamase-like domain located in its C-terminus (residues 128–458), the protein also contains a previously unrecognized N-terminal domain (residues 1–127) resembling those found in steroid isomerases and polyketide cyclases [25]. In addition, two molecules of clavulanic acid were found to be bound non-covalently to Cpe when crystals of the protein were soaked in a solution of the metabolite during structural studies [25]. The first molecule (CA-1) was positioned in an active site pocket lined by residues (His88, Ser173, Thr209, Ser234, Ser278,Met383, Phe374, Ala376 and Phe385) from both the N- and the C-terminal domains, whereas the second molecule (CA-2) bound to a mostly hydrophobic cleft at the interface of the two domains via weak ionic interactions [25].…”

“…In addition, two molecules of clavulanic acid were found to be bound non-covalently to Cpe when crystals of the protein were soaked in a solution of the metabolite during structural studies [25]. The first molecule (CA-1) was positioned in an active site pocket lined by residues (His88, Ser173, Thr209, Ser234, Ser278,Met383, Phe374, Ala376 and Phe385) from both the N- and the C-terminal domains, whereas the second molecule (CA-2) bound to a mostly hydrophobic cleft at the interface of the two domains via weak ionic interactions [25]. It was also shown that apart from Ser 173 , Ser 234 and Ser 378 , other residues from Cpe or its N-terminal domain are not essential for its in vitro esterase activity.…”