Ryan P. Sullivan

Cloning, characterization, and mutational analysis of a highly active and stable l-arabinitol 4-dehydrogenase from Neurospora crassa

An NAD+-dependent l-arabinitol 4-dehydrogenase (LAD, EC 1.1.1.12) from Neurospora crassa was cloned and expressed in Escherichia coli and purified to homogeneity. The enzyme was a homotetramer and contained two Zn2+ ions per subunit, displaying similar characteristics to medium-chain sorbitol dehydrogenases (SDHs). High enzymatic activity was observed for substrates l-arabinitol, adonitol, and xylitol and no activity for d-mannitol, d-arabinitol, or d-sorbitol. The enzyme showed strong preference for NAD+ but also displayed a very low yet detectable activity with NADP+. Mutational analysis of residue F59, the single different substrate-binding residue between LADs and d-SDHs, failed to confer the enzyme the ability to accept d-sorbitol as a substrate, suggesting that the amino acids flanking the active site cleft may be responsible for the different activity and affinity patterns between LADs and SDHs. This enzyme should be useful for in vivo and in vitro production of xylitol and ethanol from l-arabinose.

Structure and Engineering of l-Arabinitol 4-Dehydrogenase from Neurospora crassa

L-arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD(+) reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 A resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP(+) as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD+ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD+-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP+ was increased by 2.5 × 104-fold, whereas the cofactor preference toward NADP+ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.

Color-flow duplex screening for upper extremity proximity injuries: A low-yield strategy for therapeutic intervention

BACKGROUND Although the incidence of injury to the upper extremity screened with angiography as a result of proximity penetrating trauma is similar to that of the lower extremity, intervention rates seem to be higher. However, studies evaluating the incidence of injury as a result of proximity penetrating trauma have primarily focused on the lower extremity. This study shows the incidence and clinical significance of vascular injury as a result of proximity trauma to the upper extremity in a large cohort of patients screened with color-flow duplex. MATERIALS AND METHODS A retrospective study was conducted from January 1, 2005 to January 1, 2012 on all patients undergoing color-flow duplex as a result of proximity penetrating trauma to the upper extremity. Data on injury location, mechanism, associated extremity and nonextremity injuries, and use and results of color-flow duplex were recorded and analyzed. RESULTS A total of 341 patients were identified who underwent color-flow duplex because of proximity penetrating trauma to the upper extremity. Injuries occurred in 370 extremities, with 253 located in the upper arm and 117 in the forearm. Overall, 18 (4.9%) injuries were identified on screening duplex ultrasound, of which 12 (3.2%) were arterial and 5 (1.4%) were venous. The therapeutic intervention rate for detected injuries to the upper arm was 1.6% (4/253), whereas no injuries of the forearm were identified that necessitated intervention. CONCLUSIONS Although color-flow duplex is an inexpensive and noninvasive means of detecting injuries as a result of proximity penetrating trauma, screening upper extremity wounds is unlikely to detect clinically significant arterial injuries in need of therapeutic intervention. Venous injuries in the form of deep venous thromboses were detected in only 1.4% of patients. These findings suggest that screening for proximity penetrating trauma of the upper extremity is unlikely to detect injuries at a rate that would prove cost-effective on formal decision analysis.