Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability and remarkable reactivity towards the degradation of the synthetic polyester, polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.

SUMMARY Human monoclonal antibodies (mAbs) 447-52D and 537-10D, both coded by the VH3 gene and specific for the third variable region (V3) of the HIV-1 gp120, were found to share antigen binding structural elements including an elongated CDR H3 forming main-chain interactions with the N-terminus of the V3 crown. However, water-mediated hydrogen bonds and a unique cation-π sandwich stacking allow 447-52D to be broadly reactive with V3 containing both the GPGR and GPGQ crown motifs, while the deeper binding pocket, and a buried Glu in the binding site of 537-10D limit its reactivity to only V3 containing the GPGR motif. Our results suggest that the design of immunogens for anti-V3 antibodies should avoid the Arg at the V3 crown, as GPGR-containing epitopes appear to select for B cells making antibodies of narrower specificity than V3 that carry Gln at this position.

The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria. To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli. Purified rHetR was unstable and degraded easily in solution. Phenylmethanesulfonyl f luoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR. Dansyl f luoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS. The labeling of rHetR with phenylmethanesulfonyl f luoride and DnsF was at the same site of rHetR and required Ca 2؉ . S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized. Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity. DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced. The labeled peptide matches a sequence from HetR. These results show that HetR is a protease.

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

Summary In the present study, PeSTZ1, a cysteine‐2/histidine‐2‐type zinc finger transcription factor, was isolated from the desert poplar, Populus euphratica, which serves as a model stress adaptation system for trees. PeSTZ1 was preferentially expressed in the young stems and was significantly up‐regulated during chilling and freezing treatments. PeSTZ1 was localized to the nucleus and bound specifically to the PeAPX2 promoter. To examine the potential functions of PeSTZ1, we overexpressed it in poplar 84K hybrids (Populus alba × Populus glandulosa), which are known to be stress‐sensitive. Upon exposure to freezing stress, transgenic poplars maintained higher photosynthetic activity and dissipated more excess light energy (in the form of heat) than wild‐type poplars. Thus, PeSTZ1 functions as a transcription activator to enhance freezing tolerance without sacrificing growth. Under freezing stress, PeSTZ1 acts upstream of ASCORBATE PEROXIDASE2 (PeAPX2) and directly regulates its expression by binding to its promoter. Activated PeAPX2 promotes cytosolic APX that scavenges reactive oxygen species (ROS) under cold stress. PeSTZ1 may operate in parallel with C‐REPEAT‐BINDING FACTORS to regulate COLD‐REGULATED gene expression. Moreover, PeSTZ1 up‐regulation reduces malondialdehyde and ROS accumulation by activating the antioxidant system. Taken together, these results suggested that overexpressing PeSTZ1 in 84K poplar enhances freezing tolerance through the modulation of ROS scavenging via the direct regulation of PeAPX2 expression.