The purpose of this study was to investigate the proposed mechanism of catalysis for the 6-hydroxynicotinate 3-monooxygenase (NicC). NicC catalyzes the decarboxylative hydroxylation of 6-hydroxynicotinate (6-HNA) to 2,5- dihydroxypyridine (2,5-DHP). Recently the enzyme has been structurally and functionally characterized and a putative active site model has been reported. Based on studies that suggest that the enzyme utilizes acid-base catalysis, a possible catalytic mechanism for the enzyme had been proposed. The putative active site model reveals two amino acids His211 and His302 as possible candidates that could play the role of a general base in the proposed mechanism. The importance of these two histidines in catalysis has been investigated through kinetic analysis of the His211Ala and His302Ala variants of the enzyme. The steady state kinetics for the H211A variant of NicC was determined as kcat = 0.9 ± 0.02 s-1 and kcat/KM = (4.5 ± 1.6) X 104 M-1s-1 and for the H302A variant of NicC was determined as kcat = 2.3 ± 0.1 s-1 and kcat/KM = (2.9 ± 1.5) X 104 M-1s-1. The H211A variant showed only a 2- fold decrease and the H302A showed negligible decrease in catalytic efficiency when compared to wild-type NicC. These results suggest that none of these histidines are catalytically important and thus can be ruled out as the general base in the proposed mechanism. Kinetic inhibition studies were conducted on NicC with 6-ClNA (a substrate analog of NicC). The inhibition showed that 6-ClNA is a very weak non- competitive inhibitor of NicC with a Ki of 17.8 +/- 1.7 mM


Snider, Mark


Biochemistry and Molecular Biology


Medicine and Health Sciences

Publication Date


Degree Granted

Bachelor of Arts

Document Type

Senior Independent Study Thesis



© Copyright 2016 Alvi N. Sakib