Elizabeth A. Burks

Secretory leukocyte protease inhibitor: inhibition of human immunodeficiency virus-1 infection of monocytic THP-1 cells by a newly cloned protein

The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI), to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been reported previously and has led to the suggestion that SLPI may be partially responsible for the low oral transmission rate of HIV-1. However, results contradictory to these findings have also been published. These discrepancies can be attributed to a number of factors ranging from the variability of macrophage susceptibility to HIV infection to the quality of commercially available preparations of SLPI. To resolve these differences and to study further the potential anti-HIV-1 activity of SLPI, the purified and re-folded protein, expressed from a synthetic gene, was examined using human monocytic THP-1 cells. This newly cloned SLPI reduced HIV-1(Ba-L) infection in differentiated THP-1 cells, in contrast to the results observed when using commercially available preparations of SLPI. Interestingly, while the two proteins displayed different anti-HIV effects they had comparable anti-protease activity. The identification of the THP-1 cell line as a system that supports HIV replication, which can be inhibited by a preparation of SLPI now available in large quantities, sets the stage for a thorough investigation of the molecular and structural basis for the anti-HIV activity of SLPI.

Kinetic and Structural Characterization of a Heterohexamer 4- Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily †

4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three alphabeta dimers. Like the P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the beta-alpha-beta building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.

Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway.

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

Identification and characterization of new family members in the tautomerase superfamily: Analysis and implications

Tautomerase superfamily members are characterized by a β-α-β building block and a catalytic amino terminal proline. 4-Oxalocrotonate tautomerase (4-OT) and malonate semialdehyde decarboxylase (MSAD) are the title enzymes of two of the five known families in the superfamily. Two recent developments in these families indicate that there might be more metabolic diversity in the tautomerase superfamily than previously thought. 4-OT homologues have been identified in three biosynthetic pathways, whereas all previously characterized 4-OTs are found in catabolic pathways. In the MSAD family, homologues have been characterized that lack decarboxylase activity, but have a modest hydratase activity using 2-oxo-3-pentynoate. This observation stands in contrast to the first characterized MSAD, which is a proficient decarboxylase and a less efficient hydratase. The hydratase activity was thought to be a vestigial and promiscuous activity. However, this recent discovery suggests that the hydratase activity might reflect a new activity in the MSAD family for an unknown substrate. These discoveries open up new avenues of research in the tautomerase superfamily.

Structural and kinetic characterization of two 4-oxalocrotonate tautomerases in Methylibium petroleiphilum strain PM1.

Methylibium petroleiphilum strain PM1 uses various petroleum products including the fuel additive methyl tert-butyl ether and straight chain and aromatic hydrocarbons as sole carbon and energy sources. It has two operons, dmpI and dmpII, that code for the enzymes in a pair of parallel meta-fission pathways. In order to understand the roles of the pathways, the 4-oxalocrotonate tautomerase (4-OT) isozyme from each pathway was characterized. Tautomerase I and tautomerase II have the lowest pairwise sequence identity (35%) among the isozyme pairs in the parallel pathways, and could offer insight into substrate preferences and pathway functions. The kinetic parameters of tautomerase I and tautomerase II were determined using 2-hydroxymuconate and 5-(methyl)-2-hydroxymuconate. Both tautomerase I and tautomerase II process the substrates, but with different efficiencies. Crystal structures were determined for both tautomerase I and tautomerase II, at 1.57 and 1.64Å resolution, respectively. The backbones of tautomerase I and tautomerase II are highly similar, but have distinct active site environments. The results, in combination with those for other structurally and kinetically characterized 4-OT isozymes, suggest that tautomerase I catalyzes the tautomerization of both 2-hydroxymuconate and alkyl derivatives, whereas tautomerase II might specialize in other aromatic hydrocarbon metabolites.