Structural Basis for the Strict Substrate Selectivity of the Mycobacterial Hydrolase LipW

The complex life cycle of <i>Mycobacterium tuberculosis</i> requires diverse energy mobilization and utilization strategies facilitated by a battery of lipid metabolism enzymes. Among lipid metabolism enzymes, the Lip family of mycobacterial serine hydrolases is essential to lipid scavenging, metabolic cycles, and reactivation from dormancy. On the basis of the homologous rescue strategy for mycobacterial drug targets, we have characterized the three-dimensional structure of full length LipW from <i>Mycobacterium marinum</i>, the first structure of a catalytically active Lip family member. LipW contains a deep, expansive substrate-binding pocket with only a narrow, restrictive active site, suggesting tight substrate selectivity for short, unbranched esters. Structural alignment reinforced this strict substrate selectivity of LipW, as the binding pocket of LipW aligned most closely with the bacterial acyl esterase superfamily. Detailed kinetic analysis of two different LipW homologues confirmed this strict substrate selectivity, as each homologue selected for unbranched propionyl ester substrates, irrespective of the alcohol portion of the ester. Using comprehensive substitutional analysis across the binding pocket, the strict substrate selectivity of LipW for propionyl esters was assigned to a narrow funnel in the acyl-binding pocket capped by a key hydrophobic valine residue. The polar, negatively charged alcohol-binding pocket also contributed to substrate orientation and stabilization of rotameric states in the catalytic serine. Together, the structural, enzymatic, and substitutional analyses of LipW provide a connection between the structure and metabolic properties of a Lip family hydrolase that refines its biological function in active and dormant tuberculosis infection

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin

Overall structure of <i>B. pseudomallei</i> BpaA third head domain.

<p>A. Monomeric structure of the third head domain of BpaA B. Quaternary trimeric structure of third head domain of BpaA C. Trimer top down view D. Bottom-up view.</p

Sequence motifs in the third head domain of <i>B. pseudomallei</i> BpaA Trimeric Autotransporter Adhesin (TAA).

<p>Coloring indicates sequence motifs identified by the daTAA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Szczesny1" target="_blank">[3]</a> which is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone-0012803-g001" target="_blank">Figure 1</a>. A) BpaA third head domain monomer B) BpaA third head domain trimer C) FGG motif D) HANS motif. Hydrogen bonds shown as dashed lines.</p

Surface and interior structure of <i>B. pseudomallei</i> BpaA third head domain.

<p>A. One monomer of the BpaA head is sown in green ribbons with side chains shown in stick representation, while the other two monomers of the trimer are shown as a translucent molecular surface rendering in gray. B. Electrostatic surface potential mapped onto a molecular surface rendering of the third BpaA head domain. Blue indicates regions of positive charge and red indicates regions of negative charge.</p

Comparison of architecture of TAA head domain sequence motifs.

<p>Trimeric structures are shown for the TAA head domains of BadA from <i>Bartonella henselae</i> (PDB ID 3D9X <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Szczesny2" target="_blank">[11]</a>), HiaBD2 (PDB ID 3EMF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Meng1" target="_blank">[9]</a>), KG1-W3 (PDB ID 3EMI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Meng1" target="_blank">[9]</a>), and HiaBD1 (PDB ID 1S7M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Yeo1" target="_blank">[10]</a>), from <i>Haemophilus influenzae</i>, YadA from <i>Yersinia enterocolitica</i> (PDB ID 1P9H <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Nummelin1" target="_blank">[7]</a>), and BpaA from <i>B. pseudomallei</i> (PDB ID 3LAA). Trp-ring motifs are shown in yellow, GIN motifs are shown in light blue, neck regions are shown in brown, left handed β-roll Ylhead repeats are shown in magenta, FGG motifs are shown in dark blue, HANS motifs are shown in green, HIN2 motifs are shown in orange, and other regions in gray.</p

Primary structure and domain annotation of BpaA from <i>Burkholderia pseudomallei</i>.

<p>A. Domain architecture of the <i>B. pseudomallei</i> BpaA trimeric autotransporter adhesin (TAA). The BpaA TAA features an N-terminal secretion sequence, four head domains, and a C-terminal membrane anchored domain. The residues between head domains are identified as regions of low complexity and the region between the fourth head domain and the membrane anchored domain is likely to be a coiled-coil. The third head domain contains numerous sequence motifs identified by the domain annotation of Trimeric Autotransporter Adhesins (daTAA) server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012803#pone.0012803-Szczesny1" target="_blank">[3]</a> B. Multiple sequence alignment of four head domains of <i>B. pseudomallei</i> BpaA The four head domains were aligned according to their sequence and motifs identified by the daTAA are indicated.</p

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

<div><p>Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.</p></div

Comparison of PDB 4HW3 and MBP-MCL1 with fragment 4.

<p>The structure of MBP-MCL1 with fragment <b>4</b> (yellow) determined to 2.4 Å (blue) overlaid with the structure of MCL1 171–323 determined at 2.4 Å (PDB ID 4HW3, gray). The carboxylic acid of 4HW3 adopts multiple conformations depending on the chain; only chain A is shown for clarity.</p

MCL1 ligands used in co-crystallization experiments.

<p>MCL1 ligands used in co-crystallization experiments.</p