Steven W. Polyak

Biotin Protein Ligase from Saccharomyces cerevisiae THE N-TERMINAL DOMAIN IS REQUIRED FOR COMPLETE ACTIVITY

Catalytically active biotin protein ligase fromSaccharomyces cerevisiae (EC 6.3.4.15) was overexpressed inEscherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. colistrains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.

Structure, function and selective inhibition of bacterial acetyl-coa carboxylase

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.

Selective inhibition of biotin protein ligase from Staphylococcus aureus.

Background: Inhibitors of biotin protein ligase potentially represent a new antibiotic class. Results: Biotin triazoles inhibit the BPL from Staphylococcus aureus but not the human homologue. Conclusion: Our most potent inhibitor shows cytotoxicity against S. aureus but not cultured mammalian cells. Significance: This is the first report demonstrating selective inhibition of BPL. There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

5-benzylidenerhodanine and 5-benzylidene-2-4-thiazolidinedione based antibacterials.

Herein we outline the antibacterial activity of amino acid containing thiazolidinediones and rhodanines against Gram-positive bacteria Staphylococcus aureus ATCC 31890, Staphylococcus epidermidis and Bacillus subtilis ATCC 6633. The rhodanine derivatives were generally more active than the analogous thiazolidinediones. Compounds of series 5 showed some selectivity for Bacillus subtilis ATCC 6633, the extent of which is enhanced by the inclusion of a non-polar amino acid at the 5-position of the core thiazolidinediones and rhodanines scaffolds. SAR data of series 8 demonstrated improved activity against the clinically more significant Staphylococci with selectivity over Bacillus subtilis ATCC 6633 induced by introduction of a bulky aryl substituent at the 5-position of the core scaffolds.

Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency.

The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPLs from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.

Holocarboxylase synthetase: correlation of protein localisation with biological function.

Holocarboxylase synthetase (HCS) governs the cellular fate of the essential micronutrient biotin (Vitamin H or B7). HCS is responsible for attaching biotin onto the biotin-dependent enzymes that reside in the cytoplasm and mitochondria. Evidence for an alternative role, viz the regulation of gene expression, has also been reported. Recent immunohistochemical studies reported HCS is primarily nuclear, inconsistent with the location of HCS activity. Improved understanding of biotin biology demands greater knowledge about HCS. Here, we investigated the localisation of HCS and its isoforms. Three variants were observed that differ at the N-terminus. All HCS isoforms were predominantly non-nuclear, consistent with the distribution of biotin protein ligase activity. Unlike the longer constructs, the Met(58) isoform was also detected in the nucleus--a novel observation suggesting shuttling activity between nucleus and cytoplasm. We resolved that the previous controversies in the literature are due to specificity and detection limitations that arise when using partially purified antibodies.

A novel molecular mechanism to explain biotin-unresponsive holocarboxylase synthetase deficiency.

Biotin (vitamins H and B7) is an important micronutrient as defects in its availability, metabolism or adsorption can cause serious illnesses, especially in the young. A key molecule in the biotin cycle is holocarboxylase synthetase (HLCS), which attaches biotin onto the biotin-dependent enzymes. Patients with congenital HLCS deficiency are prescribed oral biotin supplements that, in most cases, reverse the clinical symptoms. However, some patients respond poorly to biotin therapy and have an extremely poor long-term prognosis. Whilst a small number of mutations in the HLCS gene have been implicated, the molecular mechanisms that lead to the biotin-unresponsive phenotype are not understood. To improve our understanding of HLCS, limited proteolysis was performed together with yeast two-hybrid analysis. A structured domain within the N-terminal region that contained two missense mutations was identified in patients who were refractory to biotin therapy, namely p.L216R and p.L237P. Genetic studies demonstrated that the interaction between the enzyme and the protein substrate was disrupted by mutation. Further dissection of the binding mechanism using surface plasmon resonance demonstrated that the mutations reduced affinity for the substrate through a >15-fold increase in dissociation rate. Together, these data provide the first molecular explanation for HLCS-deficient patients that do not respond to biotin therapy.

Biotin Analogues with Antibacterial Activity Are Potent Inhibitors of Biotin Protein Ligase

There is a desperate need to develop new antibiotic agents to combat the rise of drug-resistant bacteria, such as clinically important Staphylococcus aureus. The essential multifunctional enzyme, biotin protein ligase (BPL), is one potential drug target for new antibiotics. We report the synthesis and characterization of a series of biotin analogues with activity against BPLs from S. aureus, Escherichia coli, and Homo sapiens. Two potent inhibitors with K i < 100 nM were identified with antibacterial activity against a panel of clinical isolates of S. aureus (MIC 2-16 μg/mL). Compounds with high ligand efficiency and >20-fold selectivity between the isozymes were identified and characterized. The antibacterial mode of action was shown to be via inhibition of BPL. The bimolecular interactions between the BPL and the inhibitors were defined by surface plasmon resonance studies and X-ray crystallography. These findings pave the way for second-generation inhibitors and antibiotics with greater potency and selectivity.

Biotin biosynthesis in Mycobacterium tuberculosis : physiology, biochemistry and molecular intervention

Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug‐resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo‐ and apo‐forms of SaBPL using X‐ray crystallography. We also present small‐angle X‐ray scattering data of SaBPL in complex with its biotin‐carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl‐5′‐AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery.