Steven L. Roderick

A left-handed parallel beta helix in the structure of UDP-N-acetylglucosamine acyltransferase.

UDP-N-acetylglucosamine 3-O-acyltransferase (LpxA) catalyzes the transfer of (R)-3-hydroxymyristic acid from its acyl carrier protein thioester to UDP-N-acetylglucosamine. LpxA is the first enzyme in the lipid A biosynthetic pathway and is a target for the design of antibiotics. The x-ray crystal structure of LpxA has been determined to 2.6 angstrom resolution and reveals a domain motif composed of parallel β strands, termed a left-handed parallel β helix (LβH). This unusual fold displays repeated violations of the protein folding constraint requiring right-handed crossover connections between strands of parallel β sheets and may be present in other enzymes that share amino acid sequence homology to the repeated hexapeptide motif of LpxA.

Structure of human phosphatidylcholine transfer protein in complex with its ligand

Phosphatidylcholines (PtdChos) comprise the most common phospholipid class in eukaryotic cells. In mammalian cells, these insoluble molecules are transferred between membranes by a highly specific phosphatidylcholine transfer protein (PC-TP) belonging to the steroidogenic acute regulatory protein related transfer (START) domain superfamily of hydrophobic ligand-binding proteins. The crystal structures of human PC-TP in complex with dilinoleoyl-PtdCho or palmitoyl-linoleoyl-PtdCho reveal that a single well-ordered PtdCho molecule occupies a centrally located tunnel. The positively charged choline headgroup of the lipid engages in cation–π interactions within a cage formed by the faces of three aromatic residues. These binding determinants and those for the phosphoryl group may be exposed to the lipid headgroup at the membrane–water interface by a conformational change involving the amphipathic C-terminal helix and an Ω-loop. The structures presented here provide a basis for rationalizing the specificity of PC-TP for PtdCho and may identify common features used by START proteins to bind their hydrophobic ligands.

The Active Site of O-Acetylserine Sulfhydrylase Is the Anchor Point for Bienzyme Complex Formation with Serine Acetyltransferase

The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the alpha-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: Evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O‐acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O‐acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5′‐phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C‐terminal decapeptide bind to the α‐carboxyl subsite of O‐acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O‐acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O‐acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.

Design of O-acetylserine sulfhydrylase inhibitors by mimicking Nature

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing l-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico, docked into OASS active site using GOLD, and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and, to a lesser extent, at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases.

Mycothiol is the predominant low‐molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine–glucosamine–inositol (Cys–GlcN–Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl–CoA. MshD consists of two tandem‐repeated domains, each exhibiting the Gcn5‐related N‐acetyltransferase (GNAT) fold. These two domains superimpose with a root‐mean‐square deviation of 1.7 Å over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C‐terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl–CoA bound to the N‐terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys–GlcN–Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products.

The biosynthesis of UDP‐GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA‐dependent acetylation of GlcN‐1‐PO4 to form GlcNAc‐1‐PO4 and its subsequent condensation with UTP to form pyrophosphate and UDP‐GlcNAc. As a metabolite, UDP‐GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl‐CoA, CoA/GlcN‐1‐PO4, and desulpho‐CoA/GlcNAc‐1‐PO4. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2‐amino group of GlcN‐1‐PO4 is positioned in proximity to the acetyl‐CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

Structure of the lac operon galactoside acetyltransferase.

The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

The regulation of enzyme activity through the transient formation of multiprotein assemblies plays an important role in the control of biosynthetic pathways. One of the first regulatory complexes to be discovered was cysteine synthase (CS), formed by the pyridoxal 5′-phosphate-dependent enzyme O-acetylserine sulfhydrylase (OASS) and serine acetyltransferase (SAT). These enzymes are at the branch point of the sulfur, carbon, and nitrogen assimilation pathways. Understanding the mechanism of complex formation helps to clarify the role played by CS in the regulation of sulfur assimilation in bacteria and plants. To this goal, stopped-flow fluorescence spectroscopy was used to characterize the interaction of SAT with OASS, at different temperatures and pH values, and in the presence of the physiological regulators cysteine and bisulfide. Results shed light on the mechanism of complex formation and regulation, so far poorly understood. Cysteine synthase assembly occurs via a two-step mechanism involving rapid formation of an encounter complex between the two enzymes, followed by a slow conformational change. The conformational change likely results from the closure of the active site of OASS upon binding of the SAT C-terminal peptide. Bisulfide, the second substrate and a feedback inhibitor of OASS, stabilizes the CS complex mainly by decreasing the back rate of the isomerization step. Cysteine, the product of the OASS reaction and a SAT inhibitor, slightly affects the kinetics of CS formation leading to destabilization of the complex.

Crystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonate

Streptococcus pneumoniae, a ubiquitous gram‐positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 Å and in complex with DPM·Mg2+ in the active‐site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary‐complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK‐binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency.