ABSTRACT
Staphylococcus aureus is an opportunistic pathogen that rapidly acquires resistance to frontline antibiotics. The characterization of novel protein targets from this bacterium is thus an important step towards future therapeutic strategies. Here, the crystal structure of an amidohydrolase, SACOL0085, from S. aureus COL is described. SACOL0085 is a member of the M20D family of peptidases. Unlike other M20D peptidases, which are either monomers or dimers, SACOL0085 adopts a butterfly-shaped homotetrameric arrangement with extensive intersubunit interactions. Each subunit of SACOL0085 contains two Mn(2+) ions at the active site. A conserved cysteine residue at the active site distinguishes M20D peptidases from other M20 family members. This cysteine, Cys103, serves as bidentate ligand coordinating both Mn(2+) ions in SACOL0085.
Subject(s)
Amidohydrolases/chemistry , Bacterial Proteins/chemistry , Methicillin-Resistant Staphylococcus aureus/enzymology , Amino Acid Sequence , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Molecular Sequence Data , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target.
Subject(s)
Dihydrodipicolinate Reductase/chemistry , Dihydrodipicolinate Reductase/metabolism , NADP/metabolism , Staphylococcus aureus/enzymology , Amino Acid Sequence , Crystallography, X-Ray , Dihydrodipicolinate Reductase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Substrate SpecificityABSTRACT
Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn(2+)-dependent dipeptidase. The crystal structure of this protein in the Mn(2+)-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Manganese/metabolism , Metalloproteases/chemistry , Metalloproteases/metabolism , Staphylococcus aureus/enzymology , Bacterial Proteins/genetics , Catalytic Domain/genetics , Chromatography, Gel , Crystallography, X-Ray , Dipeptidases/chemistry , Dipeptidases/genetics , Dipeptidases/metabolism , Kinetics , Metalloproteases/genetics , Protein Structure, SecondaryABSTRACT
Lysine biosynthesis is crucial for cell-wall formation in bacteria. Enzymes involved in lysine biosynthesis are thus potential targets for anti-microbial therapeutics. Dihydrodipicolinate synthase (DHDPS) catalyzes the first step of this pathway. Unlike its homologues, Staphylococcus aureus DHDPS is a dimer both in solution and in the crystal and is not feedback inhibited by lysine. The crystal structure of S. aureus DHDPS in the free and substrate bound forms provides a structural rationale for its catalytic mechanism. The structure also reveals unique conformational features of the S. aureus enzyme that could be crucial for the design of specific non-competitive inhibitors.
