New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (βα) 8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (βα) 8 barrel as an inherently evolvable protein scaffold.HisA | TrpF | adaptive evolution | enzyme performance threshold

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.Similar to other invertebrates, insects lack an adaptive immune system and rely solely on their innate immune mechanisms to fight against invading microorganisms (1-3). These defense mechanisms are mediated by hemocytes (e.g. phagocytosis and encapsulation), fat body (e.g. induced synthesis of antimicrobial peptides), and plasma factors (e.g. hemolymph coagulation and melanization). Accumulating evidence indicates that a complex serine proteinase network in insect hemolymph coordinates some of these responses (4). The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5, 6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.Although the physiological importance of the proPO activation in insect immune system has been appreciated for many years, the molecular characterization of the cascade components was reported only recently. We isolated three proPOactivating proteinases from cuticular extract or hemolymph of M. sexta prepupae, and they all required a protein cofactor for proPO activation (7-10). Similar results were obtained from the beetles Holotrichia diomphalia and Tenebrio molitor (11-13). In contrast, the silkworm proPO-activating enzyme does not appear to need any auxiliary factor for proPO activation (14). Limited by substrate availability, current biochemical research is focused on the components at the end of th...

Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.

Asian cotton (Gossypium arboreum L.) was once widely cultivated in China. It has also been a valuable source of genetic variation in modern cotton improvement. In this study, the genetic diversity of selected G. arboreum accessions collected from different regions of China was evaluated by microsatellite (simple sequence repeats, SSRs) analysis. Of the 358 microsatellite markers analyzed, 74 primer pairs detected 165 polymorphic DNA fragments among 39 G. arboreum accessions examined. Twelve accessions could be fingerprinted with one or more SSR markers. With the exception of two accessions, DaZiJie and DaZiMian, genetic similarity coefficients among all accessions ranged from 0.58 to 0.87 suggesting high level of genetic variation in the G. arboreum collections. The UPGMA dendrogram constructed from genetic similarity coefficients revealed positive correlation between cluster groupings and geographic distances. In addition, comparison of the microsatellite amplification profiles of the diploid G. arboreum and tetraploid Gossypium hirsutum L. found that size distribution of amplified products in G. arboreum was dispersive and that of G. hirsutum was relatively concentrated. The information on the genetic diversity and SSR fingerprinting from this study is useful for developing mapping populations for constructing diploid cotton genetic linkage map and tagging economically important traits.

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Background: HisA catalyzes a ring-opening isomerization reaction in histidine biosynthesis. Results: Catalytic residues and conformational changes upon substrate binding are clarified by structures, kinetics, and mutagenesis. Conclusion: Closing of active site loops in HisA brings the substrate into a product-like conformation before catalysis. Significance: This exemplifies coupled conformational changes in a (␤␣) 8 barrel enzyme and its substrate and clarifies the mechanistic cycle of HisA.