Identification and analysis of a hydrazide-degrading enzyme
from mycobacteria
Ken-Ichi OINUMA
Department of Bacteriology, Osaka City University Graduate School ofMedicine
Research aims
Hydrazine and its derivatives constitute an important group of compounds withtremendous industrial value, being used as dyes, adhesives, pharmaceuticals, herbicides, orprecursors of these products. Although these compounds are known to exist in nature as
secondary metabolites of microorganisms, their biosynthetic and degradation pathways arelargely unknown. Acylated derivatives of hydrazine are called hydrazides. Several reports
have described hydrazide-degrading enzymes. For example, Toida reported in the 1960s that
Mycobacterium avium, a non-tubercular mycobacterium, produces an enzyme named
hydrazidase that hydrolyzes the anti-tubercular agent isoniazid (INH)[1] (Fig. 1). Anotherreport from the 1970s describes INH-hydrolyzing activity in Mycobacterium smegmatis[2].
These enzymes are important research subjects not only because of their possible
involvement in INH resistance in mycobacteria but also because of their industrial value asbiocatalysts. In the last several decades, however, no progress has been made in these studies,and the identity of these enzymes remains to be elucidated. In this study, we aimed to isolate
and identify a hydrazidase from a mycobacterium and reveal its characteristics anddistribution in nature.

Fig. 1. Isoniazid-hydrolyzing reaction catalyzed by hydrazidase.
Methods
M. smegmatis MC2155, a frequently used model strain for studying mycobacteria, waschosen as the subject for our study. An M. avium clinical isolate, obtained from the Osaka
City University Hospital, and Mycobacterium bovis BCG Pasteur were used to investigate
the distribution of INH-hydrolyzing activity among mycobacteria. These bacteria weregrown using Middlebrook 7H9 or M9-based liquid media at 37 °C with shaking.

INH-hydrolyzing activity was measured by monitoring formation of isonicotinic acid using
an ultra performance liquid chromatography system equipped with an ACQUITY UPLC
BEH Amide column (2.1 × 100 mm) (Waters). Columns manufactured by GE Healthcare
were used for protein purification. Identification of the purified enzyme was performed bypeptide mass fingerprinting based on matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry analysis following trypsin digestion.
Results
We first performed an enzyme assay using 7H9-grown M. smegmatis MC2155 cells, which
demonstrated the INH-hydrolyzing activity of this strain. We then tested the dependence of
this enzyme activity on the composition of the growth media. Although the activity was low
in 7H9-grown cells, it increased dramatically after incubating cells for 8 hours in anINH-containing synthetic medium (Fig. 2). Addition of glycerol and MgSO4 to the synthetic
media resulted in higher activity. We tried to purify the enzyme from cells grown under
optimal INH-producing conditions. Using a combination of ammonium sulfate precipitationand several different strategies of column chromatography, we succeeded in purifying theenzyme to homogeneity (Fig. 3). Peptide mass fingerprinting analysis of the purified
material revealed that the enzyme was an amidase named PzaA that has been known tohydrolyze pyrazinamide and nicotinamide [3]. These results demonstrate, for the first time,that PzaA is involved in INH degradation by M. smegmatis.
To investigate the distribution of hydrazidase among other mycobacteria, we measured theINH-hydrolyzing activity of an M. avium clinical isolate and Mycobacterium bovis BCG
Pasteur. Both strains showed similar levels of activity as M. smegmatis MC2155. We thentried to identify the enzyme using a BLAST search with the PzaA amino acid sequence as
query. Although several amidase-like gene products were found, with sequence identities ashigh as 30-35% with PzaA, we could not conclusively identify the INH-hydrolyzing
enzymes due to lack of strong candidates with prominently high homology.
Fig. 2. Effect of synthetic media additives
on the INH-hydrolyzing activity of M.
smegmatis. M. smegmatis MC2155 cells
grown in 7H9 were transferred to synthetic
media
containing
theindicatedcomponents and incubated for 8 hours.
The activities of resting cells werenormalized by optical density. Bars
indicate standard deviations (n = 3).

Fig. 3. Purification of the INH-hydrolyzing enzyme from M. smegmatis MC2155. An imageof a CBB-stained SDS-PAGE gel is shown. The arrow indicates the purified enzyme.
Conclusion
The INH-hydrolyzing enzyme of M. smegmatis has been identified as PzaA, revealing apreviously unknown secondary function of this amidase as a hydrazidase. This is the firstsuccessful identification of a mycobacterial hydrazidase. Although we assume that enzymes
similar to PzaA underlie the hydrazidase activities seen in other mycobacterial species, we
could not identify them by homology searches of available genome sequences. Aiming at
medical and zymological applications, we plan to continue this research, by including
characterization of PzaA and identification of INH-hydrolyzing enzymes from othermycobacteria.
References
1) Toida, I. (1962) Isoniazid-hydrolyzing enzyme of mycobacteria. Am. Rev. Respir. Dis. 85:
720-726.
2) Fishbain, D., Ling, G., and Kushner, D. J. (1972) Isoniazid metabolism and binding bysensitive and resistant strains of Mycobacterium smegmatis. Can. J. Microbiol. 18:
783-792.
3) Boshoff, H. I. M. and Mizrahi, V. (1998) Purification, gene cloning, targeted knockout,overexpression, and biochemical characterization of the major pyrazinamidase from
Mycobacterium smegmatis. J. Bacteriol. 180: 5809-5814.